TW202128997A - Oligonucleotides for reduction of pd-l1 expression - Google Patents

Oligonucleotides for reduction of pd-l1 expression Download PDF

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TW202128997A
TW202128997A TW109135754A TW109135754A TW202128997A TW 202128997 A TW202128997 A TW 202128997A TW 109135754 A TW109135754 A TW 109135754A TW 109135754 A TW109135754 A TW 109135754A TW 202128997 A TW202128997 A TW 202128997A
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oligonucleotide
conjugate
twenty
nucleosides
antisense oligonucleotide
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TW109135754A
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TWI794662B (en
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萊可 佩得森
海珊 傑凡巴克特
梅蘭妮 傑克羅特
索倫 歐特森
蘇法隆尼 盧安賽
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瑞士商赫孚孟拉羅股份公司
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Abstract

The present invention relates to antisense oligonucleotides that are capable of reducing expression of PD-L1 in a target cell. The oligonucleotides hybridize to PD-L1 mRNA. The present invention further relates to conjugates of the oligonucleotide and pharmaceutical compositions and methods for treatment of viral liver infections such as HBV, HCV and HDV; a parasite infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis or liver cancer or metastases in the liver using the oligonucleotide.

Description

用於降低PD-L1表現之寡核苷酸Oligonucleotides for reducing PD-L1 performance

本發明係關於與程式性死亡配體-1 (PD-L1)互補以降低肝中之PD-L1表現之寡核苷酸(寡聚物)。本發明亦係關於緩解由肝感染或肝癌引起之T細胞耗竭之方法。相關感染係慢性HBV、HCV及HDV及諸如瘧疾及弓蟲症等寄生蟲感染(例如由瘧原蟲屬(Plasmodium )、尤其物種間日瘧原蟲(P.vivax )、三日瘧原蟲(P. malariae )及惡性瘧原蟲(P. falciparum )之原生動物引起)。The present invention relates to oligonucleotides (oligomers) that are complementary to programmed death ligand-1 (PD-L1) to reduce the expression of PD-L1 in the liver. The present invention also relates to methods for alleviating T cell depletion caused by liver infection or liver cancer. Related infections are chronic HBV, HCV and HDV, and disorders such as malaria and Toxoplasmosis parasitic infections (e.g., by a Plasmodium (Plasmodium), in particular between species Plasmodium vivax (P. vivax), Plasmodium malariae ( P. malariae ) and Plasmodium falciparum ( P. falciparum ) caused by protozoa).

由程式性死亡-1 (PD-1)受體及其配體PD-L1 (或B7-H1或CD274)組成之共刺激路徑已知直接促成T細胞耗竭，從而使得在慢性肝感染期間缺乏病毒控制。PD-1路徑亦在自體免疫性中發揮一定作用，此乃因此路徑遭到破壞之小鼠發生自體免疫疾病。 已展示，阻斷PD-1與PD-L1之間之相互作用之抗體會增強T細胞反應、尤其CD8+細胞毒性T細胞之反應(參見Barber等人，2006 Nature第439卷第682頁及Maier等人，2007 J. Immunol.第178卷第2714頁)。 WO 2006/042237闡述藉由評價腫瘤中之PD-L1 (B7-H1)表現來診斷癌症之方法且建議向患者遞送干擾PD-1/PD-L1相互作用之藥劑。干擾劑可為抗體、抗體片段、siRNA或反義寡核苷酸。並未提供該等干擾劑之具體實例且並未提及任何慢性肝感染。 亦在(例如) WO 2005/007855、WO 2007/084865及US 8,507,663中揭示使用雙鏈RNA (dsRNA、RNAi或siRNA)分子來達成RNA干擾調介之PD-L1抑制。該等專利並未闡述至肝之靶向遞送。 Dolina等人， 2013 Molecular Therapy-Nucleic Acids, 2 e72闡述在活體內將靶向PD-L1之siRNA分子遞送至庫弗氏細胞(Kupffer cell)，由此增強MCMV感染小鼠中之NK及CD8+ T細胞清除。此論文斷定，遞送至肝細胞之靶向PD-L1之siRNA分子不能有效地增強CD8+ T細胞效應物功能。 siRNA方式顯著不同於單鏈反義寡核苷酸方式，此乃因生物分佈及作用模式極為不同。如Xu等人，2003 Biochem. Biophys. Res .Comm.第306卷第712-717頁中所闡述，反義寡核苷酸及siRNA對mRNA中之靶位點具有不同偏好。 WO2016/138278闡述使用兩種或更多種在5’端連接之單鏈反義寡核苷酸來抑制免疫檢查點(包含PD-L1)。本申請案並未提及HBV或至肝之靶向遞送。 本發明目標  本發明鑑別在肝臟細胞中(在實質細胞(例如肝細胞)及非實質細胞(例如庫弗氏細胞及肝竇狀隙內皮細胞(LSEC))中)極有效降低PD-L1 mRNA之新穎寡核苷酸及寡核苷酸偶聯物。藉由降低或沉默PD-L1，寡核苷酸及寡核苷酸偶聯物降低PD-1介導之抑制且由此促進耗竭T細胞之免疫刺激。緩解肝之慢性病原性感染中之T細胞耗竭使得重獲免疫控制且在肝之慢性病原性感染期間降低血液中之病毒抗原含量。天然殺手(NK)細胞及天然殺手T (NKT)細胞亦可由本發明之寡核苷酸及寡核苷酸偶聯物活化。 寡核苷酸偶聯物使得肝臟細胞中之PD-L1發生局部降低且由此降低與PD-L1之全身性消耗有關之自體免疫副效應(例如肺炎、非病毒性肝炎及結腸炎)的風險。The costimulatory pathway consisting of the programmed death-1 (PD-1) receptor and its ligand PD-L1 (or B7-H1 or CD274) is known to directly contribute to T cell exhaustion, resulting in a lack of virus during chronic liver infection control. The PD-1 pathway also plays a role in autoimmunity, and this is because mice whose pathways are destroyed develop autoimmune diseases. It has been shown that antibodies that block the interaction between PD-1 and PD-L1 can enhance T cell responses, especially CD8+ cytotoxic T cell responses (see Barber et al., 2006 Nature Vol. 439, page 682 and Maier et al. Human, 2007 J. Immunol. Vol. 178, p. 2714). WO 2006/042237 describes a method for diagnosing cancer by evaluating the performance of PD-L1 (B7-H1) in tumors and suggests the delivery of drugs that interfere with PD-1/PD-L1 interaction to patients. Interfering agents can be antibodies, antibody fragments, siRNA or antisense oligonucleotides. No specific examples of these interfering agents are provided and no chronic liver infections are mentioned. For example, WO 2005/007855, WO 2007/084865 and US 8,507,663 disclose the use of double-stranded RNA (dsRNA, RNAi or siRNA) molecules to achieve RNA interference-mediated PD-L1 inhibition. These patents do not describe targeted delivery to the liver. Dolina et al., 2013 Molecular Therapy-Nucleic Acids, 2 e72 described the delivery of siRNA molecules targeting PD-L1 to Kupffer cells in vivo, thereby enhancing NK and CD8+ T in MCMV-infected mice. Cell clearance. This paper concluded that PD-L1 targeted siRNA molecules delivered to hepatocytes cannot effectively enhance CD8+ T cell effector functions. The siRNA method is significantly different from the single-stranded antisense oligonucleotide method because of the extremely different biodistribution and mode of action. As explained in Xu et al., 2003 Biochem. Biophys. Res. Comm. Vol. 306, pages 712-717, antisense oligonucleotides and siRNA have different preferences for target sites in mRNA. WO2016/138278 describes the use of two or more single-stranded antisense oligonucleotides linked at the 5'end to suppress immune checkpoints (including PD-L1). This application does not mention HBV or targeted delivery to the liver. The objective of the present invention The present invention identifies that it is extremely effective in reducing PD-L1 mRNA in liver cells (in parenchymal cells (e.g., hepatocytes) and non-parenchymal cells (e.g., Kouffer’s cells and hepatic sinusoidal endothelial cells (LSEC)). Novel oligonucleotides and oligonucleotide conjugates. By reducing or silencing PD-L1, oligonucleotides and oligonucleotide conjugates reduce PD-1-mediated inhibition and thereby promote immune stimulation that depletes T cells. Relieve the depletion of T cells in the chronic pathogenic infection of the liver to regain immune control and reduce the level of viral antigens in the blood during the chronic pathogenic infection of the liver. Natural killer (NK) cells and natural killer T (NKT) cells can also be activated by the oligonucleotides and oligonucleotide conjugates of the present invention. Oligonucleotide conjugates cause local reduction of PD-L1 in liver cells and thereby reduce the autoimmune side effects (such as pneumonia, non-viral hepatitis and colitis) related to the systemic consumption of PD-L1 risk.

本發明係關於靶向能夠調節PD-L1表現之核酸且治療或預防與PD-L1功能相關之疾病之寡核苷酸或其偶聯物。寡核苷酸或寡核苷酸偶聯物可尤其用於治療針對傳染原之免疫反應已耗竭之疾病。 因此，在第一態樣中，本發明提供包括長度為10至30個核苷酸且與PD-L1靶核酸具有至少90%互補性之鄰接核苷酸序列之寡核苷酸。寡核苷酸可為較佳地具有間隙聚體設計之反義寡核苷酸。較佳地，寡核苷酸能夠藉由裂解靶核酸來抑制PD-L1表現。裂解較佳係經由核酸酶招募來達成。 在另一態樣中，寡核苷酸偶聯至至少一個靶向去唾液酸醣蛋白受體之偶聯物部分(例如包括至少一個N-乙醯基半乳糖胺(GalNAc)部分之偶聯物部分)。偶聯物部分及寡核苷酸可藉由連接體、尤其生物可裂解連接體連接至一起。 在另一態樣中，本發明提供包括本發明之寡核苷酸或寡核苷酸偶聯物及醫藥上可接受之稀釋劑、載劑、鹽及/或佐劑之醫藥組合物。 在另一態樣中，本發明提供降低表現PD-L1之靶細胞中之PD-L1表現之方法(活體內或活體外方法)，其係藉由向該細胞投與有效量之本發明之寡核苷酸或組合物來達成。 在另一態樣中，本發明提供用於恢復針對病毒或寄生蟲之免疫性之寡核苷酸、寡核苷酸偶聯物或醫藥組合物。 在另一態樣中，本發明提供用作藥劑之寡核苷酸、寡核苷酸偶聯物或醫藥組合物。 在另一態樣中，本發明提供治療或預防疾病、病症或功能障礙之方法，其係藉由向患有或易患該疾病、病症或功能障礙、尤其選自病毒性肝感染或寄生蟲感染之疾病之個體投與治療或預防有效量之本發明寡核苷酸來達成。 在另一態樣中，本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物係用於治療或預防病毒性肝感染(例如HBV、HCV及HDV)或寄生蟲感染(例如瘧疾、弓蟲症、利什曼病(leishmaniasis)及錐蟲病)或肝癌或肝中轉移。The present invention relates to oligonucleotides or conjugates thereof that target nucleic acids capable of regulating the expression of PD-L1 and treat or prevent diseases related to PD-L1 function. Oligonucleotides or oligonucleotide conjugates are particularly useful for treating diseases in which the immune response to infectious agents has been exhausted. Therefore, in the first aspect, the present invention provides an oligonucleotide comprising a contiguous nucleotide sequence that is 10 to 30 nucleotides in length and has at least 90% complementarity with the PD-L1 target nucleic acid. The oligonucleotide may be an antisense oligonucleotide preferably with a gapmer design. Preferably, the oligonucleotide can inhibit PD-L1 performance by cleaving the target nucleic acid. Cleavage is preferably achieved through nuclease recruitment. In another aspect, the oligonucleotide is coupled to at least one conjugate moiety that targets the asialoglycoprotein receptor (e.g., a coupling that includes at least one N-acetylgalactosamine (GalNAc) moiety)物parts). The conjugate portion and the oligonucleotide can be linked together by a linker, especially a biocleavable linker. In another aspect, the present invention provides a pharmaceutical composition comprising the oligonucleotide or oligonucleotide conjugate of the present invention and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. In another aspect, the present invention provides a method for reducing PD-L1 expression in a target cell expressing PD-L1 (in vivo or in vitro method) by administering an effective amount of the present invention to the cell Oligonucleotide or composition to achieve. In another aspect, the present invention provides oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for restoring immunity against viruses or parasites. In another aspect, the present invention provides oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for use as medicaments. In another aspect, the present invention provides methods for the treatment or prevention of diseases, disorders or dysfunctions, which are obtained by treating or predisposing to the diseases, disorders or dysfunctions, especially selected from viral liver infections or parasites. The subject of the infected disease is achieved by administering a therapeutically or preventively effective amount of the oligonucleotide of the present invention. In another aspect, the oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions of the present invention are used to treat or prevent viral liver infections (such as HBV, HCV and HDV) or parasitic infections (such as Malaria, toxoplasmosis, leishmaniasis (leishmaniasis and trypanosomiasis) or liver cancer or metastasis to the liver.

定義寡核苷酸 如熟習此項技術者通常所理解，本文所用之術語「寡核苷酸」定義為包括兩個或更多個共價連接之核苷之分子。該等共價結合之核苷亦可稱為核酸分子或寡聚物。寡核苷酸通常係在實驗室中藉由固相化學合成且隨後加以純化來製得。在提及寡核苷酸之序列時，可提及共價連接之核苷酸或核苷之核鹼基部分或其修飾之序列或順序。本發明寡核苷酸係人工製得，且係以化學方式合成，並通常進行純化或分離。本發明寡核苷酸可包括一或多個經修飾核苷或核苷酸。反義寡核苷酸 本文所用之術語「反義寡核苷酸」定義為能夠藉由雜交至靶核酸、尤其靶核酸上之鄰接序列來調節靶基因表現之寡核苷酸。反義寡核苷酸在本質上並非雙鏈且由此並非siRNA。較佳地，本發明之反義寡核苷酸係單鏈。鄰接核苷酸序列 術語「鄰接核苷酸序列」係指寡核苷酸中與靶核酸互補之區域。該術語可在本文中與術語「鄰接核鹼基序列」及術語「寡核苷酸基序序列」互換使用。在一些實施例中，寡核苷酸之所有核苷酸構成鄰接核苷酸序列。在一些實施例中，寡核苷酸包括鄰接核苷酸序列且可視情況包括其他核苷酸(例如可用於將官能基連接至鄰接核苷酸序列之核苷酸連接體區)。核苷酸連接體區可或可不與靶核酸互補。核苷酸 核苷酸係寡核苷酸及多核苷酸之組成單元且出於本發明目的其包含天然及非天然核苷酸。在性質上，核苷酸(例如DNA及RNA核苷酸)包括核糖糖部分、核鹼基部分及一或多個磷酸酯基團(其不存在於核苷中)。核苷及核苷酸亦可互換地稱為「單元」或「單體」。經修飾核苷 本文所用之術語「經修飾核苷」或「核苷修飾」係指與等效DNA或RNA核苷相比藉由引入一或多個糖部分或(核)鹼基部分之修飾而加以修飾之核苷。在一較佳實施例中，經修飾核苷包括經修飾糖部分。術語經修飾核苷亦可在本文中與術語「核苷類似物」或經修飾「單元」或經修飾「單體」互換使用。經修飾核苷間鏈接 如熟習此項技術者通常所理解，術語「經修飾核苷間鏈接」定義為除磷酸二酯(PO)鏈接外之以共價方式將兩個核苷偶合至一起之鏈接。具有經修飾核苷間鏈接之核苷亦稱為「經修飾核苷酸」。在一些實施例中，與磷酸二酯鏈接相比，經修飾核苷間鏈接增加了寡核苷酸之核酸酶抗性。對於天然寡核苷酸而言，核苷間鏈接包含在毗鄰核苷之間產生磷酸二酯鍵之磷酸酯基團。經修飾核苷間鏈接尤其可用於穩定用於活體內應用之寡核苷酸，且可用於防止在本發明寡核苷酸中之DNA或RNA核苷區域中發生核酸酶裂解(例如在間隙聚體寡核苷酸之間隙區內以及在經修飾核苷之區域中)。 在一實施例中，寡核苷酸包括一或多個自天然磷酸二酯修飾成(例如)對核酸酶攻擊更具抗性之鏈接之核苷間鏈接。可藉由在血清中培育寡核苷酸或藉由使用核酸酶抗性分析(例如蛇毒磷酸二酯酶(SVPD))來測定核酸酶抗性，此兩種方法在業內已眾所周知。能夠增強寡核苷酸之核酸酶抗性之核苷間鏈接稱為核酸酶抗性核苷間鏈接。在一些實施例中，修飾寡核苷酸或其鄰接核苷酸序列中之至少50%之核苷間鏈接，舉例而言，修飾寡核苷酸或其鄰接核苷酸序列中之至少60%、例如至少70%、例如至少80或例如至少90%之核苷間鏈接。在一些實施例中，修飾寡核苷酸或其鄰接核苷酸序列之所有核苷間鏈接。應認識到，在一些實施例中，將本發明寡核苷酸連接至非核苷酸官能基(例如偶聯物)之核苷可為磷酸二酯。在一些實施例中，寡核苷酸或其鄰接核苷酸序列之所有核苷間鏈接皆係核酸酶抗性核苷間鏈接。 經修飾核苷間鏈接可選自包括硫代磷酸酯、二硫代磷酸酯及硼烷磷酸酯之組。在一些實施例中，經修飾核苷間鏈接與本發明寡核苷酸之RNaseH招募相容，例如硫代磷酸酯、二硫代磷酸酯或硼烷磷酸酯。 在一些實施例中，核苷間鏈接包括硫(S)，例如硫代磷酸酯核苷間鏈接。 硫代磷酸酯核苷間鏈接因核酸酶抗性、有益藥物動力學及製造便利性尤其有用。在一些實施例中，寡核苷酸或其鄰接核苷酸序列中之至少50%之核苷間鏈接係硫代磷酸酯，舉例而言，寡核苷酸或其鄰接核苷酸序列中之至少60%、例如至少70%、例如至少80或例如至少90%之核苷間鏈接係硫代磷酸酯。在一些實施例中，寡核苷酸或其鄰接核苷酸序列之所有核苷間鏈接係硫代磷酸酯。 在一些實施例中，寡核苷酸包括一或多個中性核苷間鏈接，尤其係選自磷酸三酯、甲基膦酸酯、MMI、醯胺-3、甲縮醛或硫代甲縮醛之核苷間鏈接。 其他核苷間鏈接揭示於WO2009/124238 (以引用方式併入本文中)中。在一實施例中，核苷間鏈接係選自WO2007/031091 (以引用方式併入本文中)中所揭示之連接體。特定而言，核苷間鏈接可選自-O-P(O)2 -O-、-O-P(O,S)-O-、-O-P(S)2 -O-、-S-P(O)2 -O-、-S-P(O,S)-O-、-S-P(S)2 -O-、-O-P(O)2 -S-、-O-P(O,S)-S-、-S-P(O)2 -S-、-O-PO(RH )-O-、O-PO(OCH3 )-O-、-O-PO(NRH )-O-、-O-PO(OCH2 CH2 S-R)-O-、-O-PO(BH3 )-O-、-O-PO(NHRH )-O-、-O-P(O)2 -NRH -、-NRH -P(O)2 -O-、-NRH -CO-O-、-NRH -CO-NRH -，及/或核苷間連接體可選自由以下組成之群：-O-CO-O-、-O-CO-NRH -、-NRH -CO-CH2 -、-O-CH2 -CO-NRH -、-O-CH2 -CH2 -NRH -、-CO-NRH -CH2 -、-CH2 -NRH CO-、-O-CH2 -CH2 -S-、-S-CH2 -CH2 -O-、-S-CH2 -CH2 -S-、-CH2 -SO2 -CH2 -、-CH2 -CO-NRH -、-O-CH2 -CH2 -NRH -CO-、-CH2 -NCH3 -O-CH2 -，其中RH 係選自氫及C1-4-烷基。 核酸酶抗性鏈接(例如硫代磷酸酯鏈接)尤其可用於能夠在與靶核酸形成雙螺旋體時招募核酸酶之寡核苷酸區域，例如用於間隙聚體之區域G或頭聚體及尾聚體之未修飾核苷區域。然而，硫代磷酸酯鏈接亦可可用於非核酸酶招募區域及/或親和力增強區域(例如用於間隙聚體之區域F及F’或頭聚體及尾聚體之經修飾核苷區域)中。 然而，每一設計區域可包括除硫代磷酸酯外之核苷間鏈接(例如磷酸二酯鏈接)，尤其在經修飾核苷(例如LNA)保護鏈接抵抗核酸酶降解之區域中。納入磷酸二酯鏈接(例如一或兩個鏈接，尤其在經修飾核苷單元之間或毗鄰該等經修飾核苷單元(通常位於非核酸酶招募區域中))可改變寡核苷酸之生物可用性及/或生物分佈-參見WO2008/113832 (以引用方式併入本文中)。 在一實施例中，寡核苷酸中之所有核苷間鏈接皆係硫代磷酸酯及/或硼烷磷酸酯鏈接。較佳地，寡核苷酸中之所有核苷間鏈接皆係硫代磷酸酯鏈接。核鹼基 術語核鹼基包含存在於核苷及核苷酸中之在核酸雜交中形成氫鍵之嘌呤(例如腺嘌呤及鳥嘌呤)及嘧啶(例如尿嘧啶、胸腺嘧啶及胞嘧啶)部分。在本發明背景中，術語核鹼基亦涵蓋經修飾核鹼基，該等經修飾核鹼基可不同於天然核鹼基，但在核酸雜交期間具有功能性。在此背景中，「核鹼基」係指天然核鹼基(例如腺嘌呤、鳥嘌呤、胞嘧啶、胸苷、尿嘧啶、黃嘌呤及次黃嘌呤)以及非天然變體。該等變體(例如)闡述於Hirao等人(2012) Accounts of Chemical Research第45卷第2055頁及Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry增刊37 1.4.1。 在一些實施例中，藉由將嘌呤或嘧啶變成經修飾嘌呤或嘧啶(例如經取代嘌呤或經取代嘧啶)來修飾核鹼基部分，例如選自異胞嘧啶、假異胞嘧啶、5-甲基胞嘧啶、5-噻唑并-胞嘧啶、5-丙炔基-胞嘧啶、5-丙炔基-尿嘧啶、5-溴尿嘧啶、5-噻唑并-尿嘧啶、2-硫代-尿嘧啶、2’硫代-胸腺嘧啶、肌苷、二胺基嘌呤、6-胺基嘌呤、2-胺基嘌呤、2,6-二胺基嘌呤及2-氯-6-胺基嘌呤之核鹼基。 核鹼基部分可藉由用於每一相應核鹼基之字母代碼(例如A、T、G、C或U)來指示，其中每一字母可視情況包含具有等效功能之經修飾核鹼基。舉例而言，在所例示寡核苷酸中，核鹼基部分係選自A、T、G、C及5-甲基胞嘧啶。視情況，對於LNA間隙聚體而言，可使用5-甲基胞嘧啶LNA核苷。經修飾寡核苷酸 術語經修飾寡核苷酸闡述包括一或多個糖修飾性核苷及/或經修飾核苷間鏈接之寡核苷酸。術語「嵌合」寡核苷酸係在文獻中用於闡述具有經修飾核苷之寡核苷酸之術語。互補性 術語「互補性」闡述核苷/核苷之沃森-克裡克(Watson-Crick)鹼基配對之能力。沃森-克裡克鹼基對係鳥嘌呤(G)-胞嘧啶(C)及腺嘌呤(A) -胸腺嘧啶(T)/尿嘧啶(U)。應理解，寡核苷酸可包括具有經修飾核鹼基之核苷，舉例而言，通常使用5-甲基胞嘧啶代替胞嘧啶，且由此術語互補性涵蓋未修飾核鹼基與經修飾核鹼基之間之沃森-克裡克鹼基配對(例如參見Hirao等人(2012) Accounts of Chemical Research第45卷第2055頁及Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry增刊37 1.4.1)。 本文所用之術語「互補%」係指核酸分子(例如寡核苷酸)中在給定位置與單獨核酸分子(例如靶核酸)中給定位置之鄰接核苷酸序列互補(亦即形成沃森-克裡克鹼基對)之核苷酸的數量(以鄰接核苷酸序列之百分比形式)。藉由以下方式來計算百分比：計數在兩個序列之間形成對之比對鹼基數(在比對靶序列5’-3’與3’-5’之寡核苷酸序列時)，除以寡核苷酸中之核苷酸總數且乘以100。在此一對比中，並不對準(形成鹼基對)之核鹼基/核苷酸可視為失配。 術語「完全互補」係指100%互補性。 下文係與靶核酸(SEQ ID NO: 772)完全互補之寡核苷酸(SEQ ID NO: 5)之一實例。 5’gcagtagagccaatta3’ (SEQ ID NO:772) 3’cgtcatctcggttaat5’ (SEQ ID NO: 5)一致性 本文所用之術語「一致性」係指核酸分子(例如寡核苷酸)中在給定位置與單獨核酸分子(例如靶核酸)中給定位置之鄰接核苷酸序列一致(亦即能夠與互補核苷形成沃森-克裡克鹼基對)之核苷酸的數量(以鄰接核苷酸序列之百分比形式)。藉由以下方式來計算百分比：計數在兩個序列(包含間隙)之間一致之比對鹼基數，除以寡核苷酸中之核苷酸總數且乘以100。百分比一致性= (匹配數× 100)/比對區域長度(含有間隙)。雜交 本文所用之術語「雜交(hybridizing或hybridizes)」應理解為兩條核酸鏈(例如寡核苷酸與靶核酸)在相對鏈上之鹼基對之間形成氫鍵，由此形成雙螺旋體。兩條核酸鏈之間之結合親和力係雜交強度。其通常係針對熔融溫度(Tm )來進行闡述，熔融溫度定義為一半寡核苷酸與靶核酸形成雙螺旋體之溫度。在生理學條件下，Tm 並不與親和力嚴格成正比(Mergny及Lacroix, 2003,Oligonucleotides 13:515-537)。標準態吉布斯自由能(Gibbs free energy) ΔG°係結合親和力之更準確代表且與反應之解離常數(Kd )以ΔG°=-RTln(Kd )形式相關，其中R係氣體常數且T係絕對溫度。因此，寡核苷酸與靶核酸之間之反應之極低ΔG°反映寡核苷酸與靶核酸之間的強雜交。ΔG°係與反應有關之能量，其中水性濃度為1M，pH為7，且溫度為37℃。寡核苷酸至靶核酸之雜交係自發反應且自發反應之ΔG°小於零。可以實驗方式藉由(例如)使用等溫滴定量熱(ITC)方法來量測ΔG°，如Hansen等人，1965,Chem. Comm. 36-38及Holdgate等人，2005,Drug Discov Today 中所闡述。熟習此項技術者應知曉，商業設備可用於ΔG°量測。亦可在數值上藉由使用最近鄰居模型(如由SantaLucia, 1998,Proc Natl Acad Sci USA. 95: 1460-1465所闡述)使用適當導出之熱動力學參數(由Sugimoto等人，1995,Biochemistry 34:11211-11216及McTigue等人，2004,Biochemistry 43:5388-5405所闡述)來估計ΔG°。為使得可藉由雜交來調節其預期核酸靶，本發明寡核苷酸以低於-10 kcal之估計ΔG°值(對於長10-30個核苷酸之寡核苷酸)雜交至靶核酸。在一些實施例中，藉由標準態吉布斯自由能ΔG°來量測雜交之程度或強度。對於長8-30個核苷酸之寡核苷酸而言，寡核苷酸可以低於-10 kcal範圍(例如低於-15 kcal、例如低於-20 kcal及例如低於-25 kcal)之估計ΔG°值雜交至靶核酸。在一些實施例中，寡核苷酸以(-10 kcal至-60 kcal、例如-12 kcal至-40 kcal、例如-15 kcal至-30 kcal或-16 kcal至-27 kcal、例如-18 kcal至-25 kcal)之估計ΔG°值雜交至靶核酸。靶核酸 根據本發明，靶核酸係編碼哺乳動物PD-L1之核酸且可為(例如)基因、RNA、mRNA及mRNA前體、成熟mRNA或cDNA序列。靶可由此稱為PD-L1靶核酸。本發明寡核苷酸可(例如)靶向哺乳動物PD-L1之外顯子區域，或可(例如)靶向PD-L1 mRNA前體之內含子區域(參見表1)。 表1：人類PD-L1外顯子及內含子 人類PD-L1 mRNA前體(SEQ ID NO 1)中之外顯子區域 人類PD-L1 mRNA前體(SEQ ID NO 1)中之內含子區域 ID 起始 末端 ID 起始 末端 e1 1 94 i1 95 5597 e2 5598 5663 i2 5664 6576 e3 6577 6918 i3 6919 12331 e4 12332 12736 i4 12737 14996 e5 14997 15410 i5 15411 16267 e6 16268 16327 i6 16328 17337 e7 17338 20064          適宜地，靶核酸編碼PD-L1蛋白、尤其哺乳動物PD-L1、例如人類PD-L1 (例如參見表2及3，其提供用於人類、猴及小鼠PD-L1之參考mRNA及mRNA前體序列)。在本發明之情形中，mRNA前體亦視為編碼蛋白質之核酸。 在一些實施例中，靶核酸係選自由以下組成之群：SEQ ID NO: 1、2及3或其天然變體(例如編碼哺乳動物PD-L1蛋白之序列)。 若在研究或診斷中採用本發明寡核苷酸，則靶核酸可為cDNA或衍生自DNA或RNA之合成核酸。 對於活體內或活體外應用而言，本發明寡核苷酸通常能夠抑制表現PD-L1靶核酸之細胞中之PD-L1靶核酸表現。本發明寡核苷酸之核鹼基之鄰接序列通常與PD-L1靶核酸互補，如橫跨寡核苷酸之長度所量測，視情況一或兩個失配除外，且視情況排除可將寡核苷酸連接至可選官能基(例如偶聯物)或其他非互補性末端核苷酸之基於核苷酸之連接體區(例如區域D’或D’’)。在一些實施例中，靶核酸可為RNA或DNA，例如信使RNA，例如成熟mRNA或mRNA前體。在一些實施例中，編碼哺乳動物PD-L1蛋白(例如人類PD-L1)之靶核酸係RNA或DNA (例如人類PD-L1 mRNA前體序列(例如揭示為SEQ ID NO 1者)或具有NCBI參考編號NM_014143之人類mRNA序列)。關於實例性靶核酸之其他資訊提供於表2及3中。 表2：各物種中之PD-L1之基因體及組合體資訊。 物種 Chr. 鏈 基因體坐標 開始 末端 組合體 mRNA 之NCBI 參考序列* 登錄號 人類 9 fwd 5450503 5470566 GRCh38:CM000671.2 NM_014143 食蟹猴 15    73560846 73581371 GCF_000364345.1 XM_005581779 小鼠 19 fwd 29367455 29388095 GRCm38:CM001012.2 NM_021893 Fwd =正向鏈。基因體坐標提供mRNA前體序列(基因體序列)。NCBI參考提供mRNA序列(cDNA序列)。 *國家生物技術資訊中心(National Center for Biotechnology Information)參考序列資料庫係參考序列(包含基因體、轉錄物及蛋白質)之綜合性、整合、非冗餘、充分注釋集合。其寄存於www.ncbi.nlm.nih.gov/refseq。 表3：各物種中之PD-L1之序列細節。 物種 RNA 類型 長度 (nt) SEQ ID NO 人類 mRNA前體 20064 1 食蟹猴 mRNA前體GCF ref 20261 2 食蟹猴 mRNA前體，內部 20340 3 小鼠 mRNA前體 20641 4 靶序列 本文所用之術語「靶序列」係指存在於靶核酸中之包括與本發明寡核苷酸互補之核鹼基序列的核苷酸序列。在一些實施例中，靶序列係由靶核酸上與本發明寡核苷酸之鄰接核苷酸序列互補之區域組成。在一些實施例中，靶序列長於單一寡核苷酸之互補序列，且可(例如)代表靶核酸中可由若干本發明寡核苷酸靶向之較佳區域。 靶序列可為靶核酸之子序列。 在一些實施例中，子序列係選自由a1-a149 (參見表4)組成之群之序列。在一些實施例中，子序列係選自由人類PD-L1 mRNA外顯子(例如選自由以下組成之群之PD-L1人類mRNA外顯子：e1、e2、e3、e4、e5、e6及e7 (參見上表1))組成之群之序列。 在一些實施例中，子序列係選自由人類PD-L1 mRNA內含子(例如選自由以下組成之PD-L1人類mRNA內含子群：i1、i2、i3、i4、i5及i6 (參見上表1))組成之群之序列。 本發明寡核苷酸包括與靶核酸(例如靶核酸之子序列，例如本文所闡述之靶序列)互補或雜交之鄰接核苷酸序列。 寡核苷酸包括具有至少8個核苷酸之與存在於靶核酸分子中之靶序列互補或雜交之鄰接核苷酸序列。鄰接核苷酸序列(及由此靶序列)包括至少8個鄰接核苷酸，例如9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個鄰接核苷酸，例如12-25個鄰接核苷酸，例如14-18個鄰接核苷酸。靶細胞 本文所用之術語「靶細胞」係指表現靶核酸之細胞。在一些實施例中，靶細胞可為活體內或活體外靶細胞。在一些實施例中，靶細胞係哺乳動物細胞，例如齧齒類動物細胞(例如小鼠細胞或大鼠細胞)或靈長類動物細胞(例如猴細胞或人類細胞)。 在較佳實施例中，靶細胞表現PD-L1 mRNA (例如PD-L1 mRNA前體或PD-L1成熟mRNA)。PD-L1 mRNA之多腺苷酸尾通常忽視反義寡核苷酸靶向。天然變體 術語「天然變體」係指PD-L1基因或轉錄物之如下變體：其與靶核酸源自相同基因座，但可(例如)因產生多種編碼相同胺基酸之密碼子之基因代碼之簡並性或因mRNA前體之替代剪接或存在多型性(例如單一核苷酸多型性)而有所不同，且係指等位基因變體。基於寡核苷酸之充分互補序列之存在，本發明寡核苷酸可由此靶向靶核酸及其天然變體。 在一些實施例中，天然變體與哺乳動物PD-L1靶核酸(例如選自由SEQ ID NO 1、2及3組成之群之靶核酸)具有至少95% (例如至少98%或至少99%)之同源性。 已知PD-L1基因中之諸多單一核苷酸多型性，例如揭示於下表中者(人類mRNA前體起始/參考序列係SEQ ID NO 2) 變體名稱 變體等位基因 次要等位基因 次要等位基因頻率 SEQ ID NO: 1 上之起點 rs73397192 G/A A 0.10 2591 rs12342381 A/G G 0.12 308 rs16923173 G/A A 0.13 14760 rs2890658 C/A A 0.16 14628 rs2890657 G/C C 0.21 2058 rs3780395 A/G A 0.21 14050 rs147367592 AG/- - 0.21 13425 rs7023227 T/C T 0.22 6048 rs2297137 G/A A 0.23 15230 rs1329946 G/A A 0.23 2910 rs5896124 -/G G 0.23 2420 rs61061063 T/C C 0.23 11709 rs1411263 T/C C 0.23 8601 rs59906468 A/G G 0.23 15583 rs6476976 T/C T 0.24 21012 rs35744625 C/A A 0.24 3557 rs17804441 T/C C 0.24 7231 rs148602745 C/T T 0.25 22548 rs4742099 G/A A 0.25 20311 rs10815228 T/C C 0.25 21877 rs58817806 A/G G 0.26 20769 rs822342 T/C T 0.27 3471 rs10481593 G/A A 0.27 7593 rs822339 A/G A 0.28 2670 rs860290 A/C A 0.28 2696 rs822340 A/G A 0.28 2758 rs822341 T/C T 0.28 2894 rs12002985 C/G C 0.28 6085 rs822338 C/T C 0.28 1055 rs866066 C/T T 0.28 451 rs6651524 A/T T 0.28 8073 rs6415794 A/T A 0.28 8200 rs4143815 G/C C 0.28 17755 rs111423622 G/A A 0.28 24096 rs6651525 C/A A 0.29 8345 rs4742098 A/G G 0.29 19995 rs10975123 C/T T 0.30 10877 rs2282055 T/G G 0.30 5230 rs4742100 A/C C 0.30 20452 rs60520638 -/TC TC 0.30 9502 rs17742278 T/C C 0.30 6021 rs7048841 T/C T 0.30 10299 rs10815229 T/G G 0.31 22143 rs10122089 C/T C 0.32 13278 rs1970000 C/A C 0.32 14534 rs112071324 AGAGAG/- AGAGAG 0.33 16701 rs2297136 G/A G 0.33 17453 rs10815226 A/T T 0.33 9203 rs10123377 A/G A 0.36 10892 rs10123444 A/G A 0.36 11139 rs7042084 G/T G 0.36 7533 rs10114060 G/A A 0.36 11227 rs7028894 G/A G 0.36 10408 rs4742097 C/T C 0.37 5130 rs1536926 G/T G 0.37 13486 rs1411262 C/T T 0.39 8917 rs7041009 G/A A 0.45 12741 表現調節 本文所用之術語「表現調節」欲理解為關於寡核苷酸改變PD-L1量(與投與寡核苷酸之前之PD-L1量相比)之能力之概括性術語。或者，可藉由參照對照實驗來測定表現調節。通常應理解，對照係使用鹽水組合物治療或處理之個體或靶細胞或使用非靶寡核苷酸(模擬)治療或處理之個體或靶細胞。然而，亦可標準護理治療個體。 一類調節係寡核苷酸能夠(例如)藉由降解mRNA或阻斷轉錄來抑制、下調、降低、阻抑、去除、停止、阻斷、預防、減弱、減小、避免或終止PD-L1表現。另一類調節係寡核苷酸能夠(例如)藉由修復剪接位點或防止剪接或去除或阻斷抑制機制(例如微RNA阻遏)來恢復、增加或增強PD-L1表現。高親和力修飾性核苷 高親和力修飾性核苷係在納入寡核苷酸中時會增強寡核苷酸對其互補靶之親和力(例如如藉由熔融溫度(Tm )所量測)之經修飾核苷酸。本發明之高親和力修飾性核苷較佳地使得每一經修飾核苷之熔融溫度增加+0.5℃至+12℃、更佳地+1.5℃至10℃及最佳地+3℃至+8℃。業內已知諸多高親和力修飾性核苷且包含(例如)許多2’取代核苷以及鎖核酸(LNA) (例如參見Freier & Altmann；Nucl.  Acid Res., 1997, 25, 4429-4443及Uhlmann；Curr.  Opinion in Drug Development, 2000, 3(2), 293-213)。糖修飾 本發明寡聚物可包括一或多個具有經修飾糖部分(亦即與DNA及RNA中所發現之核糖糖部分相比之糖部分修飾)之核苷。 已製備諸多具有核糖糖部分修飾之核苷，其目的主要在於改良寡核苷酸之某些性質(例如親和力及/或核酸酶抗性)。 該等修飾包含核糖環結構(例如)藉由使用以下結構進行代替來修飾者：己糖環(HNA)；或通常在核糖環上之C2碳與C4碳之間具有雙自由基橋之雙環(LNA)；或通常在C2碳與C3碳之間缺乏鍵之未連接核糖環(例如UNA)。其他糖修飾性核苷包含(例如)雙環己糖核酸(WO2011/017521)或三環核酸(WO2013/154798)。經修飾核苷亦包含糖部分經非糖部分代替之核苷，例如在肽核酸(PNA)或嗎啉基核酸之情形下。 糖修飾亦包含經由將核糖環上之取代基改變成除氫或DNA及RNA核苷中天然發現之2’-OH基團外之基團而進行之修飾。可(例如)在2’、3’、4’或5’位置引入取代基。具有經修飾糖部分之核苷亦包含2’修飾核苷，例如2’取代核苷。實際上，高度關注研發2’取代核苷，且已發現諸多2’取代核苷在納入寡核苷酸中時具有有益性質(例如增強之核苷抗性及增強之親和力)。2’ 修飾核苷 . 2’糖修飾性核苷係在2’位具有除H或-OH外之取代基(2’取代核苷)或包括2’連接雙自由基之核苷，且包含2’取代核苷及LNA (2’ - 4’雙自由基橋接)核苷。舉例而言，2’修飾糖可向寡核苷酸提供增強之結合親和力及/或增加之核酸酶抗性。2’取代修飾性核苷之實例係2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA (MOE)、2’-胺基-DNA、2’-氟-RNA及2’-F-ANA核苷。關於其他實例，請參見(例如) Freier & Altmann；Nucl.  Acid Res., 1997, 25, 4429-4443及Uhlmann；Curr.  Opinion in Drug Development, 2000, 3(2), 293-213及Deleavey及Damha, Chemistry and Biology 2012, 19, 937。下文闡釋一些2’取代修飾性核苷。

Figure 02_image003
鎖核酸核苷 (LNA) 。  LNA核苷係在核苷酸之核糖糖環之C2’與C4’之間包括連接體基團(稱為雙基或橋)之經修飾核苷。該等核苷在文獻中亦稱為橋接核酸或雙環核酸(BNA)。 在一些實施例中，本發明寡聚物之經修飾核苷或LNA核苷具有式I或II之一般結構：
Figure 02_image005
或
Figure 02_image007
式I                        式II 其中W係選自-O-、-S-、-N(Ra )-、-C(Ra Rb )-，例如在一些實施例中係-O-； B指定核鹼基或經修飾核鹼基部分； Z指定至毗鄰核苷之核苷間鏈接或5'-末端基團； Z*指定至毗鄰核苷之核苷間鏈接或3'-末端基團； X指定選自由以下組成之列表之基團：-C(Ra Rb )-、-C(Ra )=C(Rb )-、-C(Ra )=N-、-O-、-Si(Ra )2 -、-S-、-SO2 -、-N(Ra )-及＞C=Z 在一些實施例中，X係選自由以下組成之群：-O-、-S-、NH-、NRa Rb 、-CH2 -、CRa Rb 、-C(=CH2 )-及-C(=CRa Rb )- 在一些實施例中，X係-O-。 Y指定選自由以下組成之群之基團：-C(Ra Rb )-、-C(Ra )=C(Rb )-、-C(Ra )=N-、-O-、-Si(Ra )2 -、-S-、-SO2 -、-N(Ra )-及＞C=Z 在一些實施例中，Y係選自由以下組成之群：-CH2 -、-C(Ra Rb )-、-CH2 CH2 -、-C(Ra Rb )-C(Ra Rb )-、-CH2 CH2 CH2 -、-C(Ra Rb )C(Ra Rb )C(Ra Rb )-、-C(Ra )=C(Rb )-及-C(Ra )=N- 在一些實施例中，Y係選自由以下組成之群：-CH2 -、-CHRa -、-CHCH3 -、CRa Rb - 或-X-Y-一起指定二價連接體基團(亦稱為自由基)，一起指定由1、2、3或4個選自由以下組成之群之基團/原子組成之二價連接體基團：-C(Ra Rb )-、-C(Ra )=C(Rb )-、-C(Ra )=N-、-O-、-Si(Ra )2 -、-S-、-SO2 -、-N(Ra )-及＞C=Z， 在一些實施例中，-X-Y-指定選自由以下組成之群之雙自由基：-X-CH2 -、-X-CRa Rb -、-X-CHRa- 、-X-C(HCH3 )- 、-O-Y-、-O-CH2 -、-S-CH2 -、-NH-CH2 -、-O-CHCH3 -、-CH2 -O-CH2 、-O-CH(CH3 CH3 )-、-O-CH2 -CH2 -、OCH2 -CH2 -CH2 -、-O-CH2 OCH2 -、-O-NCH2 -、-C(=CH2 )-CH2 -、-NRa -CH2 -、N-O-CH2 、-S-CRa Rb -及-S-CHRa -。 在一些實施例中，-X-Y-指定-O-CH2 -或-O-CH(CH3 )-。 其中Z係選自-O-、-S-及-N(Ra )-， 且Ra 及(在存在時) Rb 各自獨立地係選自氫、視情況經取代之C1-6 -烷基、視情況經取代之C2-6 -烯基、視情況經取代之C2-6 -炔基、羥基、視情況經取代之C1-6 -烷氧基、C2-6 -烷氧基烷基、C2-6 -烯基氧基、羧基、C1-6 -烷氧基羰基、C1-6 -烷基羰基、甲醯基、芳基、芳基氧基-羰基、芳基氧基、芳基羰基、雜芳基、雜芳基氧基-羰基、雜芳基氧基、雜芳基羰基、胺基、單-及二(C1-6 -烷基)胺基、胺甲醯基、單-及二(C1-6 -烷基)-胺基-羰基、胺基-C1-6 -烷基-胺基羰基、單-及二(C1-6 -烷基)胺基-C1-6 -烷基-胺基羰基、C1-6 -烷基-羰基胺基、脲基、C1-6 -烷醯基氧基、磺醯基、C1-6 -烷基磺醯基氧基、硝基、疊氮基、硫烷基、C1-6 -硫烷基、鹵素，其中芳基及雜芳基可視情況經取代且其中兩個孿位取代基Ra 及Rb 一起可指定視情況經取代之亞甲基(=CH2 )，其中對於所有對掌性中心而言，不對稱基團可以R 或S 定向發現。 其中R1 、R2 、R3 、R5 及R5* 獨立地選自由以下組成之群：氫、視情況經取代之C1-6 -烷基、視情況經取代之C2-6 -烯基、視情況經取代之C2-6 -炔基、羥基、C1-6 -烷氧基、C2-6 -烷氧基烷基、C2-6 -烯基氧基、羧基、C1-6 -烷氧基羰基、C1-6 -烷基羰基、甲醯基、芳基、芳基氧基-羰基、芳基氧基、芳基羰基、雜芳基、雜芳基氧基-羰基、雜芳基氧基、雜芳基羰基、胺基、單-及二(C1-6 -烷基)胺基、胺甲醯基、單-及二(C1-6 -烷基)-胺基-羰基、胺基-C1-6 -烷基-胺基羰基、單-及二(C1-6 -烷基)胺基-C1-6 -烷基-胺基羰基、C1-6 -烷基-羰基胺基、脲基、C1-6 -烷醯基氧基、磺醯基、C1-6 -烷基磺醯基氧基、硝基、疊氮基、硫烷基、C1-6 -硫烷基、鹵素，其中芳基及雜芳基可視情況經取代，且其中兩個孿位取代基一起可指定側氧基、側硫基、亞胺基或視情況經取代之亞甲基。 在一些實施例中，R1 、R2 、R3 、R5 及R5* 獨立地選自C1-6 烷基(例如甲基)及氫。 在一些實施例中，R1 、R2 、R3 、R5 及R5* 皆係氫。 在一些實施例中，R1 、R2 、R3 皆係氫，且R5 及R5* 中之任一者亦係氫且R5 及R5* 中之另一者不為氫(例如C1-6 烷基，例如甲基)。 在一些實施例中，Ra 係氫或甲基。在一些實施例中，在存在時，Rb 係氫或甲基。 在一些實施例中，Ra 及Rb 中之一者或兩者係氫。 在一些實施例中，Ra 及Rb 中之一者係氫且另一者不為氫。 在一些實施例中，Ra 及Rb 中之一者係甲基且另一者係係氫。 在一些實施例中，Ra 及Rb 皆係甲基。 在一些實施例中，雙自由基-X-Y-係-O-CH2 -，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等LNA核苷揭示於WO99/014226、WO00/66604、WO98/039352及WO2004/046160 (其皆以引用方式併入本文中)中，且包含通常稱為β-D- 氧基 LNA 及α-L- 氧基 LNA 核苷者。 在一些實施例中，雙自由基-X-Y-係-S-CH2 -，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等硫代 LNA 核苷揭示於WO99/014226及WO2004/046160 (其以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-係-NH-CH2 -，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等胺基 LNA 核苷揭示於WO99/014226及WO2004/046160 (其以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-係-O-CH2 -CH2 -或-O-CH2 -CH2 - CH2 -，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等LNA核苷揭示於WO00/047599及Morita等人，Bioorganic & Med. Chem. Lett. 12 73-76 (其以引用方式併入本文中)中，且包含通常稱為2’-O-4’C-伸乙基橋接之核酸(ENA)者。 在一些實施例中，雙自由基-X-Y-係-O-CH2 -，W係O，且R1 、R2 、R3 之全部及R5 及R5* 中之一者係氫，且R5 及R5* 中之另一者不為氫(例如C1-6 烷基，例如甲基)。該等5’ 取代 LNA核苷揭示於WO2007/134181 (其以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-係-O-CRa Rb -，其中Ra 及Rb 中之一者或兩者不為氫(例如甲基)，W係O，且R1 、R2 、R3 之全部及中之一者R5 及R5* 係氫，且R5 及R5* 中之另一者不為氫(例如C1-6 烷基，例如甲基)。該等雙修飾 LNA 核苷 揭示於WO2010/077578 (其以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-指定二價連接體基團-O-CH(CH2 OCH3 )- (2’O-甲氧基乙基雙環核酸- Seth等人，2010, J. Org. Chem.第75卷(5)第1569-81頁)。在一些實施例中，雙自由基-X-Y-指定二價連接體基團-O-CH(CH2 CH3 )- (2’O-乙基雙環核酸- Seth等人，2010, J. Org. Chem.第75卷(5)第1569-81頁)。在一些實施例中，雙自由基-X-Y-係-O-CHRa -，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等6’ 取代 LNA核苷揭示於WO10036698及WO07090071 (二者皆以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-係-O-CH(CH2 OCH3 )-，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等LNA核苷在業內亦稱為環狀 MOE (cMOE)且揭示於WO07090071中。 在一些實施例中，雙自由基-X-Y-指定二價連接體基團-O-CH(CH3 )-。-呈R-或S-構形。在一些實施例中，雙自由基-X-Y-一起指定二價連接體基團-O-CH2 -O-CH2 - (Seth等人，2010, J. Org. Chem)。在一些實施例中，雙自由基-X-Y-係-O-CH(CH3 )-，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等6’甲基LNA核苷在業內亦稱為cET 核苷 ，且可為(S)cET或(R)cET立體異構體，如WO07090071 (β-D)及WO2010/036698 (α-L) (二者皆以引用方式併入本文中)中所揭示。 在一些實施例中，雙自由基-X-Y-係-O-CRa Rb -，其中Ra 或Rb 皆不為氫，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。在一些實施例中，Ra 及Rb 皆係甲基。該等6’ 二取代 LNA核苷揭示於WO 2009006478 (其以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-係-S-CHRa -，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等6’ 取代硫代 LNA核苷揭示於WO11156202 (其以引用方式併入本文中)中。在一些6’取代硫代LNA實施例中，Ra 係甲基。 在一些實施例中，雙自由基-X-Y-係-C(=CH2)-C(Ra Rb )- (例如-C(=CH2 )-CH2 -或-C(=CH2 )-CH(CH3 )-)，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。該等乙烯基碳 LNA核苷揭示於WO08154401及WO09067647 (二者皆以引用方式併入本文中)中。 在一些實施例中，雙自由基-X-Y-係-N(-ORa )- W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。在一些實施例中，Ra 係C1-6 烷基，例如甲基。該等LNA核苷亦稱為N取代LNA且揭示於WO2008/150729 (其以引用方式併入本文中)中。在一些實施例中，雙自由基-X-Y-一起指定二價連接體基團-O-NRa -CH3 - (Seth等人，2010, J. Org. Chem)。在一些實施例中，雙自由基-X-Y-係-N(Ra )-，W係O，且R1 、R2 、R3 、R5 及R5* 皆係氫。在一些實施例中，Ra 係C1-6 烷基，例如甲基。 在一些實施例中，R5 及R5* 中之一者或兩者係氫且(在取代時)R5 及R5* 中之另一者係C1-6 烷基(例如甲基)。在此一實施例中，R1 、R2 、R3 可皆係氫，且雙自由基-X-Y-可選自-O-CH2-或-O-C(HCRa )- (例如-O-C(HCH3)-)。 在一些實施例中，雙自由基係-CRa Rb -O-CRa Rb - (例如CH2 -O-CH2 -)，W係O且R1 、R2 、R3 、R5 及R5* 皆係氫。在一些實施例中，Ra 係C1-6 烷基，例如甲基。該等LNA核苷亦稱為構象限制性核苷酸(CRN)且揭示於WO2013036868 (其以引用方式併入本文中)中。 在一些實施例中，雙自由基係-O-CRa Rb -O-CRa Rb - (例如O-CH2 -O-CH2 -)，W係O且R1 、R2 、R3 、R5 及R5* 皆係氫。在一些實施例中，Ra 係C1-6 烷基，例如甲基。該等LNA核苷亦稱為COC核苷酸且揭示於Mitsuoka等人，Nucleic Acids Research 2009 37(4), 1225-1238 (其以引用方式併入本文中)中。 應認識到，除非指定，否則LNA核苷可呈β-D或α-L立體異構體形式。 LNA核苷之某些實例呈現於結構圖1中。 結構圖1
Figure 02_image009
如實例中所闡釋，在本發明之一些實施例中，寡核苷酸中之LNA核苷係β-D-氧基-LNA核苷。核酸酶調介之降解 核酸酶調介之降解係指在與互補核苷酸序列形成雙螺旋體時能夠調介此一序列之降解之寡核苷酸。 在一些實施例中，寡核苷酸可經由靶核酸之核酸酶調介之降解來發揮作用，其中本發明寡核苷酸能夠招募核酸酶、尤其內核酸酶、較佳地內核糖核酸酶(RNase) (例如RNase H)。經由核酸酶調介機制作用之寡核苷酸設計之實例係通常包括至少5或6個DNA核苷之區域且在一側或兩側側接有親和力增強性核苷(例如間隙聚體、頭聚體及尾聚體)的寡核苷酸。RNase H 活性及招募 反義寡核苷酸之RNase H活性係指其在呈與互補RNA分子之雙螺旋體時招募RNase H之能力。WO01/23613提供測定RNaseH活性之活體外方法，該等方法可用於測定招募RNaseH之能力。通常，寡核苷酸在以下情況下可視為能夠招募RNase H：在與互補靶核酸序列一起提供時，其初始速率(如以pmol/l/min形式所量測)為在使用與所測試經修飾寡核苷酸具有相同鹼基序列但僅含有DNA單體且在寡核苷酸中之所有單體間具有硫代磷酸酯鏈接之寡核苷酸且使用由WO01/23613中實例91 - 95 (以引用方式併入本文中)提供之方法時所測定初始速率的至少5% (例如至少10%或大於20%)。間隙聚體 本文所用之術語間隙聚體係指反義寡核苷酸包括招募RNase H之寡核苷酸區域(間隙)，該區域側接(5’及3’)有包括一或多個親和力增強性經修飾核苷之區域(側翼或翼)。本文闡述各種間隙聚體設計且其特徵在於能夠招募RNaseH。頭聚體及尾聚體係缺失一個側翼之能夠招募RNase H之寡核苷酸，亦即僅寡核苷酸之一端包括親和力增強性經修飾核苷。對於頭聚體而言，3’側翼缺失(亦即，5’側翼包括親和力增強性經修飾核苷)，且對於尾聚體而言，5’側翼缺失(亦即，3’側翼包括親和力增強性經修飾核苷)。LNA 間隙聚體 術語LNA間隙聚體係至少一個親和力增強性經修飾核苷係LNA核苷之間隙聚體寡核苷酸。混合翼間隙聚體 術語混合翼間隙聚體或混合側翼間隙聚體係指至少一個側翼區域包括至少一個LNA核苷及至少一個非LNA經修飾核苷(例如至少一個2’取代經修飾核苷，例如2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA (MOE)、2’-胺基-DNA、2’-氟-RNA及2’-F-ANA核苷)之LNA間隙聚體。在一些實施例中，混合翼間隙聚體具有一個僅包括LNA核苷(例如5’或3’)之側翼及包括2’取代經修飾核苷及視情況LNA核苷之另一側翼(分別3’或5’)。間隙中斷體 術語「間隙中斷體寡核苷酸」用於係指即使間隙區由非RNaseH招募性核苷(間隙中斷體核苷，E)破壞(從而間隙區包括小於5個連續DNA核苷)亦能夠維持RNAseH招募之間隙聚體。非RNaseH招募性核苷係(例如)呈3’內向構象之核苷，例如核苷之核糖糖環中C2’與C4’之間之橋係呈β構象的LNA，例如β-D-氧基LNA或ScET核苷。間隙中斷體寡核苷酸招募RNaseH之能力通常係序列或甚至化合物特異性-參見Rukov等人，2015 Nucl. Acids Res.第43卷第8476-8487頁，其揭示招募在一些情況下提供靶RNA之更特異性裂解之RNaseH之「間隙中斷體」寡核苷酸。 在一些實施例中，本發明寡核苷酸係間隙中斷體寡核苷酸。在一些實施例中，間隙中斷體寡核苷酸包括5’-側翼(F)、間隙(G)3’-側翼(F’)，其中間隙藉由非RNaseH招募核苷(間隙中斷體核苷，E)破壞，從而間隙含有至少3或4個連續DNA核苷。在一些實施例中，間隙中斷體核苷(E)係如下LNA核苷：其中核苷之核糖糖環中C2’與C4’之間之橋係呈β構象且置於間隙區內，從而間隙中斷體LNA核苷側接(5’及3’)有至少3 (5’)及3 (3’)或至少3 (5’)及4 (3’)或至少4(5’)及3(3’)個DNA核苷，且其中寡核苷酸能夠招募RNaseH。 間隙中斷體寡核苷酸可由下列各式代表： F-G-E-G-F’；尤其F1-7 -G3-4 -E1 -G3-4- F’1-7 D’-F-G-F’，尤其D’1-3 -F1-7 -G3-4 -E1 -G3-4 -F’1-7 F-G-F’-D’’，尤其F1-7 -G3-4 -E1 -G3-4 -F’1-7 -D’’1-3 D’-F-G-F’-D’’，尤其D’1-3 -F1-7 -G3-4 -E1 -G3-4 -F’1-7 -D’’1-3 其中區域D’及D’’係如「間隙聚體設計」部分中所闡述。 在一些實施例中，間隙中斷體核苷(E)係β-D-氧基LNA或ScET或結構圖1中所展示之另一β-LNA核苷。偶聯物 本文所用之術語偶聯物係指以共價方式連接至非核苷酸部分(偶聯物部分或區域C或第三區域)之寡核苷酸，其亦稱為寡核苷酸偶聯物。 本發明寡核苷酸至一或多個非核苷酸部分之偶聯可(例如)藉由影響寡核苷酸之活性、細胞分佈、細胞攝取或穩定性來改良寡核苷酸之藥理學。在一些實施例中，偶聯物部分使寡核苷酸靶向肝。同時，偶聯用於降低寡核苷酸在非靶細胞類型、組織或器官中之活性，例如脫靶活性或非靶細胞類型、組織或器官中之活性。在本發明之一實施例中，本發明之寡核苷酸偶聯物顯示與未偶聯寡核苷酸相比PD-L1在靶細胞中之改良抑制。在另一實施例中，與未偶聯寡核苷酸相比，本發明之寡核苷酸偶聯物在肝與其他器官(例如脾或腎)之間具有改良之細胞分佈(亦即，與脾或腎相比，更多偶聯寡核苷酸去往肝)。在另一實施例中，與未偶聯寡核苷酸相比，本發明之寡核苷酸偶聯物展示偶聯寡核苷酸在肝中之改良之細胞攝取。 WO 93/07883及WO2013/033230提供適宜偶聯物部分，該等專利以引用方式併入本文中。其他適宜偶聯物部分係能夠結合至去唾液酸醣蛋白受體(ASGPr)者。特定而言，三價N-乙醯基半乳糖胺偶聯物部分適於結合至ASGPr，例如參見WO2014/076196、WO2014/207232及WO 2014/179620 (以引用方式併入本文中)。偶聯物部分基本上係反義寡核苷酸偶聯物中並非由核酸構成之部分。 寡核苷酸偶聯物及其合成亦報導於以下綜合性綜述中：Manoharan，Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke編輯，第16章，Marcel Dekker, Inc., 2001及Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103，其中之每一者之全部內容皆以引用方式併入本文中。 在一實施例中，非核苷酸部分(偶聯物部分)係選自由以下組成之群：碳水化合物、細胞表面受體配體、藥物物質、激素、親脂性物質、聚合物、蛋白質、肽、毒素(例如細菌毒素)、維他命、病毒蛋白(例如衣殼)或其組合。連接體 鏈接或連接體係兩個原子之間經由一或多個共價鍵連接一個所關注化學基團或區段與另一所關注化學基團或區段之連結。偶聯物部分可直接或經由連接部分(例如連接體或結合體)連接至寡核苷酸。連接體用於以共價方式將第三區域(例如偶聯物部分，區域C)連結至與靶核酸互補之第一區域(例如寡核苷酸或鄰接核苷酸序列，區域A)。 在本發明之一些實施例中，本發明之偶聯物或寡核苷酸偶聯物可視情況包括連接體區(第二區域或區域B及/或區域Y)，該區域定位於與靶核酸互補之寡核苷酸或鄰接核苷酸序列(區域A或第一區域)與偶聯物部分(區域C或第三區域)之間。 區域B係指包括生理上不穩定鍵或由其組成之生物可裂解連接體，該生理上不穩定鍵可在哺乳動物體內通常所遇到之條件或類似於哺乳動物體內所遇到條件之條件下裂解。生理上不穩定連接體發生化學轉變(例如裂解)之條件包含化學條件，例如pH、溫度、氧化或還原條件或試劑及在哺乳動物細胞中所發現鹽濃度或類似於在哺乳動物細胞中所遇到鹽濃度之鹽濃度。哺乳動物細胞內條件亦包含在哺乳動物細胞中通常存在酶促活性(例如來自蛋白水解酶或水解酶或核酸酶)。在一實施例中，生物可裂解連接體易於發生S1核酸酶裂解。在一較佳實施例中，核酸酶易感連接體包括1至10個含有至少兩個連續磷酸二酯鏈接(例如至少3或4或5個連續磷酸二酯鏈接)之核苷(例如1、2、3、4、5、6、7、8、9或10個核苷、更佳地2至6個核苷及最佳地2至4個經連接核苷)。較佳地，核苷係DNA或RNA。含有磷酸二酯之生物可裂解連接體更詳細闡述於WO 2014/076195 (以引用方式併入本文中)中。 區域Y係指未必生物可裂解但主要用於以共價方式連結偶聯物部分(區域C或第三區域)與與靶核酸互補之寡核苷酸或鄰接核苷酸序列(區域A或第一區域)之連接體。區域Y連接體可包括諸如乙二醇、胺基酸單元或胺基烷基等重複單元之鏈結構或寡聚物。本發明之寡核苷酸偶聯物可由下列區域要素構成：A-C、A-B-C、A-B-Y-C、A-Y-B-C或A-Y-C。在一些實施例中，連接體(區域Y)係胺基烷基，例如C2 - C36胺基烷基，包含(例如) C6 - C12胺基烷基。在一較佳實施例中，連接體(區域Y)係C6胺基烷基。 治療  本文所用之術語「治療」係指治療現有疾病(例如如本文所提及之疾病或病症)或預防疾病(亦即預防性)。因此應認識到，如本文所提及之治療可在一些實施例中為預防性。 針對病原體之免疫反應之恢復  將免疫反應分成先天性免疫反應及適應性免疫反應。先天性免疫系統提供立即但非特異性之反應。適應性免疫反應係藉由先天性免疫反應活化且對特定病原體具有高度特異性。在將病原體源抗原呈遞於抗原呈遞細胞表面上時，適應性免疫反應之免疫細胞(亦即T及B淋巴球)經由其抗原特異性受體發生活化，從而產生病原性特異性免疫反應且產生免疫記憶。慢性病毒感染(例如HBV及HCV)與特徵在於病毒特異性T細胞之無反應性之T細胞耗竭有關。已充分研究T細胞耗竭，其綜述可參見(例如) Yi等人，2010 Immunology129, 474-481。慢性病毒感染亦與作為先天性免疫細胞之NK細胞之降低功能有關。增強病毒免疫反應對於慢性感染之清除較為重要。可藉由量測增殖、細胞介素分泌及細胞溶解功能來評價由T細胞及NK細胞調介之針對病原體之免疫反應之恢復(Dolina等人，2013 Molecular Therapy-Nucleic Acids, 2 e72及本文中之實例6)。 本發明的詳細敘述 本發明係關於反義寡核苷酸及其偶聯物及包括該等物質之醫藥組合物用以恢復針對感染動物、尤其人類之病原體之免疫反應的用途。本發明之反義寡核苷酸偶聯物尤其可用於抵抗感染肝之病原體、尤其慢性肝感染(例如HBV)。該等偶聯物容許靶向分佈寡核苷酸且防止靶核酸之全身性敲低。本發明寡核苷酸 本發明係關於能夠調節PD-L1表現之寡核苷酸。可藉由雜交至編碼PD-L1或涉及PD-L1調控之靶核酸來達成該調節。靶核酸可為哺乳動物PD-L1序列，例如選自由SEQ ID NO: 1、SEQ ID NO: 2及/或SEQ ID NO: 3組成之群之序列。靶核酸可為mRNA前體、mRNA或任一自支持PD-L1之表現或調控之哺乳動物細胞表現之RNA序列。 本發明寡核苷酸係靶向PD-L1之反義寡核苷酸。 在本發明之一態樣中，本發明寡核苷酸偶聯至偶聯物部分、尤其靶向去唾液酸醣蛋白受體之偶聯物部分。 在一些實施例中，本發明之反義寡核苷酸能夠藉由抑制或下調靶來調節靶表現。較佳地，該調節使得與正常靶表現程度相比抑制至少20%之表現，更佳地與正常靶表現程度相比抑制至少30%、40%、50%、60%、70%、80%或90%。較佳地，該調節使得與在藉由傳染原攻擊細胞或有機體或使用模擬傳染原攻擊之試劑(例如聚I:C或LPS)處理細胞或有機體時之表現程度相比抑制至少20%之表現，更佳地與在藉由傳染原攻擊細胞或有機體或使用模擬傳染原攻擊之試劑(例如聚I:C或LPS)處理細胞或有機體時之表現程度相比抑制至少30%、40%、50%、60%、70%、80%或90%。在一些實施例中，在活體外使用KARPAS-299或THP1細胞，本發明寡核苷酸可能夠抑制PD-L1 mRNA之表現程度至少60%或70%。在一些實施例中，在活體外使用KARPAS-299或THP1細胞，本發明化合物可能夠抑制PD-L1蛋白之表現程度至少50%。適宜地，實例提供可用於量測PD-L1 RNA之分析(例如實例1)。藉由寡核苷酸之鄰接核苷酸序列與靶核酸之間之雜交來觸發靶調節。在一些實施例中，本發明寡核苷酸在寡核苷酸與靶核酸之間包括失配。儘管具有失配，但雜交至靶核酸仍可足以展示PD-L1表現之期望調節。源自失配之降低之結合親和力可有利地藉由寡核苷酸中增加數量之核苷酸及/或寡核苷酸序列內所存在增加數量之能夠增加靶結合親和力的經修飾核苷(例如2’修飾核苷，包含LNA)來予以補償。 在一些實施例中，本發明之反義寡核苷酸能夠恢復病原體特異性T細胞。在一些實施例中，在與未處理對照或使用標準護理處理之對照相比時，本發明寡核苷酸能夠增加病原體特異性T細胞至少40%、50%、60%或70%。在一實施例中，在與未處理對照或使用標準護理處理之對照相比時，本發明之反義寡核苷酸或偶聯物能夠增加HBV特異性T細胞。適宜地，實例提供可用於量測HBV特異性T細胞之分析(例如T細胞增殖、細胞介素分泌及細胞溶解活性)。在另一實施例中，在與未處理對照或使用標準護理處理之對照相比時，本發明之反義寡核苷酸或偶聯物能夠增加HCV特異性T細胞。在另一實施例中，在與未處理對照或使用標準護理處理之對照相比時，本發明之反義寡核苷酸或偶聯物能夠增加HDV特異性T細胞。 在一些實施例中，本發明之反義寡核苷酸能夠降低動物或人類中之HBsAg含量。在一些實施例中，在與治療之前之含量相比時，本發明寡核苷酸能夠降低HBsAg含量至少40%、50%、60%或70%、更佳地至少80%、90%或95%。最佳地，本發明寡核苷酸能夠達成HBsAg在感染HBV之動物或人類中之血清轉化。 本發明之一態樣係關於包括與PD-L1靶核酸具有至少90%互補性之長度為10至30個核苷酸之鄰接核苷酸序列之反義寡核苷酸。 在一些實施例中，寡核苷酸包括與靶核酸區域至少90%互補、例如至少91%、例如至少92%、例如至少93%、例如至少94%、例如至少95%、例如至少96%、例如至少97%、例如至少98%或100%互補之鄰接序列。 在一較佳實施例中，本發明寡核苷酸或其鄰接核苷酸序列與靶核酸區域完全互補(100%互補)，或在一些實施例中可在寡核苷酸與靶核酸之間包括一或兩個失配。 在一些實施例中，寡核苷酸包括與存在於SEQ ID NO: 1或SEQ ID NO: 2中之靶核酸區域至少90%互補(例如完全(或100%)互補)之長度為10至30個核苷酸之鄰接核苷酸序列。在一些實施例中，寡核苷酸序列與存在於 SEQ ID NO: 1及SEQ ID NO: 2中之相應靶核酸區域100%互補。在一些實施例中，寡核苷酸序列與存在於SEQ ID NO: 1及SEQ ID NO: 3於中之相應靶核酸區域100%互補。 在一些實施例中，寡核苷酸或寡核苷酸偶聯物包括與相應靶核酸區域至少90%互補(例如100%互補)之長度為10至30個核苷酸之鄰接核苷酸序列，其中該鄰接核苷酸序列與選自由SEQ ID NO: 1上之位置371-3068、5467-12107及15317-19511組成之群之靶核酸的子序列互補。在另一實施例中，靶核酸之子序列係選自由以下組成之群：SEQ ID NO: 1上之位置371-510、822-1090、1992-3068、5467-5606、6470-12107、15317-15720、15317-18083、18881-19494及1881-19494。在一較佳實施例中，靶核酸之子序列係選自由以下組成之群：SEQ ID NO: 1上之位置7300-7333、8028-8072、9812-9859、11787-11873及15690-15735。 在一些實施例中，寡核苷酸或寡核苷酸偶聯物包括與存在於SEQ ID NO: 1中之相應靶核酸區域至少90%互補(例如100%互補)之長度為10至30個核苷酸之鄰接核苷酸序列，其中該靶核酸區域係選自由表4中之區域a1至a449組成之群。 表4：可使用本發明寡核苷酸靶向之SEQ ID NO 1之區域 Reg. a SEQ ID NO 1 中之位置 長度 Reg.a SEQ ID NO 1 中之位置 長度 Reg.a SEQ ID NO 1 中之位置 長度 起點 終點 起點 終點 起點 終點 a1 51 82 32 a151 6994 7020 27 a301 13092 13115 24 a2 87 116 30 a152 7033 7048 16 a302 13117 13134 18 a3 118 133 16 a153 7050 7066 17 a303 13136 13169 34 a4 173 206 34 a154 7078 7094 17 a304 13229 13249 21 a5 221 287 67 a155 7106 7122 17 a305 13295 13328 34 a6 304 350 47 a156 7123 7144 22 a306 13330 13372 43 a7 354 387 34 a157 7146 7166 21 a307 13388 13406 19 a8 389 423 35 a158 7173 7193 21 a308 13408 13426 19 a9 425 440 16 a159 7233 7291 59 a309 13437 13453 17 a10 452 468 17 a160 7300 7333 34 a310 13455 13471 17 a11 470 484 15 a161 7336 7351 16 a311 13518 13547 30 a12 486 500 15 a162 7353 7373 21 a312 13565 13597 33 a13 503 529 27 a163 7375 7412 38 a313 13603 13620 18 a14 540 574 35 a164 7414 7429 16 a314 13630 13663 34 a15 576 649 74 a165 7431 7451 21 a315 13665 13679 15 a16 652 698 47 a166 7453 7472 20 a316 13706 13725 20 a17 700 750 51 a167 7474 7497 24 a317 13727 13774 48 a18 744 758 15 a168 7517 7532 16 a318 13784 13821 38 a19 774 801 28 a169 7547 7601 55 a319 13831 13878 48 a20 805 820 16 a170 7603 7617 15 a320 13881 13940 60 a21 827 891 65 a171 7632 7647 16 a321 13959 14013 55 a22 915 943 29 a172 7649 7666 18 a322 14015 14031 17 a23 950 982 33 a173 7668 7729 62 a323 14034 14049 16 a24 984 1000 17 a174 7731 7764 34 a324 14064 14114 51 a25 1002 1054 53 a175 7767 7817 51 a325 14116 14226 111 a26 1060 1118 59 a176 7838 7860 23 a326 14229 14276 48 a27 1124 1205 82 a177 7862 7876 15 a327 14292 14306 15 a28 1207 1255 49 a178 7880 7944 65 a328 14313 14384 72 a29 1334 1349 16 a179 7964 8012 49 a329 14386 14408 23 a30 1399 1425 27 a180 8028 8072 45 a330 14462 14481 20 a31 1437 1458 22 a181 8086 8100 15 a331 14494 14519 26 a32 1460 1504 45 a182 8102 8123 22 a332 14557 14577 21 a33 1548 1567 20 a183 8125 8149 25 a333 14608 14628 21 a34 1569 1586 18 a184 8151 8199 49 a334 14646 14668 23 a35 1608 1662 55 a185 8218 8235 18 a335 14680 14767 88 a36 1677 1700 24 a186 8237 8276 40 a336 14765 14779 15 a37 1702 1721 20 a187 8299 8344 46 a337 14815 14844 30 a38 1723 1745 23 a188 8346 8436 91 a338 14848 14925 78 a39 1768 1794 27 a189 8438 8470 33 a339 14934 14976 43 a40 1820 1835 16 a190 8472 8499 28 a340 14978 15009 32 a41 1842 1874 33 a191 8505 8529 25 a341 15013 15057 45 a42 1889 1979 91 a192 8538 8559 22 a342 15064 15091 28 a43 1991 2011 21 a193 8562 8579 18 a343 15094 15140 47 a44 2013 2038 26 a194 8581 8685 105 a344 15149 15165 17 a45 2044 2073 30 a195 8688 8729 42 a345 15162 15182 21 a46 2075 2155 81 a196 8730 8751 22 a346 15184 15198 15 a47 2205 2228 24 a197 8777 8800 24 a347 15200 15221 22 a48 2253 2273 21 a198 8825 8865 41 a348 15232 15247 16 a49 2275 2303 29 a199 8862 8894 33 a349 15250 15271 22 a50 2302 2333 32 a200 8896 8911 16 a350 15290 15334 45 a51 2335 2366 32 a201 8938 8982 45 a351 15336 15369 34 a52 2368 2392 25 a202 8996 9045 50 a352 15394 15416 23 a53 2394 2431 38 a203 9048 9070 23 a353 15433 15451 19 a54 2441 2455 15 a204 9072 9139 68 a354 15453 15491 39 a55 2457 2494 38 a205 9150 9168 19 a355 15496 15511 16 a56 2531 2579 49 a206 9170 9186 17 a356 15520 15553 34 a57 2711 2732 22 a207 9188 9202 15 a357 15555 15626 72 a58 2734 2757 24 a208 9204 9236 33 a358 15634 15652 19 a59 2772 2786 15 a209 9252 9283 32 a359 15655 15688 34 a60 2788 2819 32 a210 9300 9331 32 a360 15690 15735 46 a61 2835 2851 17 a211 9339 9354 16 a361 15734 15764 31 a62 2851 2879 29 a212 9370 9398 29 a362 15766 15787 22 a63 2896 2912 17 a213 9400 9488 89 a363 15803 15819 17 a64 2915 2940 26 a214 9490 9537 48 a364 15846 15899 54 a65 2944 2973 30 a215 9611 9695 85 a365 15901 15934 34 a66 2973 2992 20 a216 9706 9721 16 a366 15936 15962 27 a67 2998 3016 19 a217 9723 9746 24 a367 15964 15985 22 a68 3018 3033 16 a218 9748 9765 18 a368 15987 16023 37 a69 3036 3051 16 a219 9767 9788 22 a369 16025 16061 37 a70 3114 3139 26 a220 9794 9808 15 a370 16102 16122 21 a71 3152 3173 22 a221 9812 9859 48 a371 16134 16183 50 a72 3181 3203 23 a222 9880 9913 34 a372 16185 16281 97 a73 3250 3271 22 a223 9923 9955 33 a373 16283 16298 16 a74 3305 3335 31 a224 9966 10007 42 a374 16305 16323 19 a75 3346 3363 18 a225 10009 10051 43 a375 16325 16356 32 a76 3391 3446 56 a226 10053 10088 36 a376 16362 16404 43 a77 3448 3470 23 a227 10098 10119 22 a377 16406 16456 51 a78 3479 3497 19 a228 10133 10163 31 a378 16494 16523 30 a79 3538 3554 17 a229 10214 10240 27 a379 16536 16562 27 a80 3576 3597 22 a230 10257 10272 16 a380 16564 16580 17 a81 3603 3639 37 a231 10281 10298 18 a381 16582 16637 56 a82 3663 3679 17 a232 10300 10318 19 a382 16631 16649 19 a83 3727 3812 86 a233 10339 10363 25 a383 16655 16701 47 a84 3843 3869 27 a234 10409 10426 18 a384 16737 16781 45 a85 3874 3904 31 a235 10447 10497 51 a385 16783 16804 22 a86 3926 3955 30 a236 10499 10529 31 a386 16832 16907 76 a87 3974 3993 20 a237 10531 10546 16 a387 16934 16965 32 a88 3995 4042 48 a238 10560 10580 21 a388 16972 17035 64 a89 4053 4073 21 a239 10582 10596 15 a389 17039 17069 31 a90 4075 4123 49 a240 10600 10621 22 a390 17072 17109 38 a91 4133 4157 25 a241 10623 10664 42 a391 17135 17150 16 a92 4158 4188 31 a242 10666 10685 20 a392 17167 17209 43 a93 4218 4250 33 a243 10717 10773 57 a393 17211 17242 32 a94 4277 4336 60 a244 10775 10792 18 a394 17244 17299 56 a95 4353 4375 23 a245 10794 10858 65 a395 17304 17344 41 a96 4383 4398 16 a246 10874 10888 15 a396 17346 17400 55 a97 4405 4446 42 a247 10893 10972 80 a397 17447 17466 20 a98 4448 4464 17 a248 10974 10994 21 a398 17474 17539 66 a99 4466 4493 28 a249 10996 11012 17 a399 17561 17604 44 a100 4495 4558 64 a250 11075 11097 23 a400 17610 17663 54 a101 4571 4613 43 a251 11099 11124 26 a401 17681 17763 83 a102 4624 4683 60 a252 11140 11157 18 a402 17793 17810 18 a103 4743 4759 17 a253 11159 11192 34 a403 17812 17852 41 a104 4761 4785 25 a254 11195 11226 32 a404 17854 17928 75 a105 4811 4858 48 a255 11235 11261 27 a405 17941 18005 65 a106 4873 4932 60 a256 11279 11337 59 a406 18007 18035 29 a107 4934 4948 15 a257 11344 11381 38 a407 18041 18077 37 a108 4955 4974 20 a258 11387 11411 25 a408 18085 18146 62 a109 4979 5010 32 a259 11427 11494 68 a409 18163 18177 15 a110 5012 5052 41 a260 11496 11510 15 a410 18179 18207 29 a111 5055 5115 61 a261 11512 11526 15 a411 18209 18228 20 a112 5138 5166 29 a262 11528 11551 24 a412 18230 18266 37 a113 5168 5198 31 a263 11570 11592 23 a413 18268 18285 18 a114 5200 5222 23 a264 11594 11634 41 a414 18287 18351 65 a115 5224 5284 61 a265 11664 11684 21 a415 18365 18395 31 a116 5286 5302 17 a266 11699 11719 21 a416 18402 18432 31 a117 5317 5332 16 a267 11721 11746 26 a417 18434 18456 23 a118 5349 5436 88 a268 11753 11771 19 a418 18502 18530 29 a119 5460 5512 53 a269 11787 11873 87 a419 18545 18590 46 a120 5514 5534 21 a270 11873 11905 33 a420 18603 18621 19 a121 5548 5563 16 a271 11927 11942 16 a421 18623 18645 23 a122 5565 5579 15 a272 11946 11973 28 a422 18651 18708 58 a123 5581 5597 17 a273 11975 11993 19 a423 18710 18729 20 a124 5600 5639 40 a274 12019 12114 96 a424 18731 18758 28 a125 5644 5661 18 a275 12116 12135 20 a425 18760 18788 29 a126 5663 5735 73 a276 12137 12158 22 a426 18799 18859 61 a127 5737 5770 34 a277 12165 12192 28 a427 18861 18926 66 a128 5778 5801 24 a278 12194 12216 23 a428 18928 18980 53 a129 5852 5958 107 a279 12218 12246 29 a429 19001 19018 18 a130 6007 6041 35 a280 12262 12277 16 a430 19034 19054 21 a131 6049 6063 15 a281 12283 12319 37 a431 19070 19092 23 a132 6065 6084 20 a282 12334 12368 35 a432 19111 19154 44 a133 6086 6101 16 a283 12370 12395 26 a433 19191 19213 23 a134 6119 6186 68 a284 12397 12434 38 a434 19215 19240 26 a135 6189 6234 46 a285 12436 12509 74 a435 19255 19356 102 a136 6236 6278 43 a286 12511 12543 33 a436 19358 19446 89 a137 6291 6312 22 a287 12545 12565 21 a437 19450 19468 19 a138 6314 6373 60 a288 12567 12675 109 a438 19470 19512 43 a139 6404 6447 44 a289 12677 12706 30 a439 19514 19541 28 a140 6449 6482 34 a290 12708 12724 17 a440 19543 19568 26 a141 6533 6555 23 a291 12753 12768 16 a441 19570 19586 17 a142 6562 6622 61 a292 12785 12809 25 a442 19588 19619 32 a143 6624 6674 51 a293 12830 12859 30 a443 19683 19739 57 a144 6679 6762 84 a294 12864 12885 22 a444 19741 19777 37 a145 6764 6780 17 a295 12886 12916 31 a445 19779 19820 42 a146 6782 6822 41 a296 12922 12946 25 a446 19822 19836 15 a147 6824 6856 33 a297 12948 12970 23 a447 19838 19911 74 a148 6858 6898 41 a298 12983 13003 21 a448 19913 19966 54 a149 6906 6954 49 a299 13018 13051 34 a449 19968 20026 59 a150 6969 6992 24 a300 13070 13090 21             在一些實施例中，寡核苷酸或鄰接核苷酸序列與靶核酸區域互補，其中靶核酸區域係選自由以下組成之群：a7、a26、a43、a119、a142、a159、a160、a163、a169、a178、a179、a180、a189、a201、a202、a204、a214、a221、a224、a226、a243、a254、a258、269、a274、a350、a360、a364、a365、a370、a372、a381、a383、a386、a389、a400、a427、a435及a438。 在一較佳實施例中，寡核苷酸或鄰接核苷酸序列與靶核酸區域互補，其中靶核酸區域係選自由以下組成之群：a160、a180、a221、a269及a360。 在一些實施例中，本發明寡核苷酸包括8至35個核苷酸(長度)或由其組成，例如長9至30、例如10至22、例如11至20、例如12至18、例如13至17或14至16個鄰接核苷酸。在一較佳實施例中，寡核苷酸包括16至20個核苷酸(長度)或由其組成。應理解，本文所給出之任一範圍皆包含範圍端點。因此，若提及寡核苷酸包含10至30個核苷酸，則包含10及30個核苷酸。 在一些實施例中，鄰接核苷酸序列包括8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個鄰接核苷酸(長度)或由其組成。在一較佳實施例中，寡核苷酸包括16、17、18、19或20個核苷酸(長度)或由其組成。 在一些實施例中，寡核苷酸或鄰接核苷酸序列包括選自由表5中所列示序列組成之群之序列或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與選自由SEQ ID NO: 5至743 (參見表5中所列示之基序序列)組成之群之序列具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與選自由SEQ ID NO: 5至743及771組成之群之序列具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與選自由以下組成之群之序列具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成：SEQ ID NO: 6、8、9、13、41、42、58、77、92、111、128、151、164、166、169、171、222、233、245、246、250、251、252、256、272、273、287、292、303、314、318、320、324、336、342、343、344、345、346、349、359、360、374、408、409、415、417、424、429、430、458、464、466、474、490、493、512、519、519、529、533、534、547、566、567、578、582、601、619、620、636、637、638、640、645、650、651、652、653、658、659、660、665、678、679、680、682、683、684、687、694、706、716、728、733、734及735。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與SEQ ID NO: 287具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與SEQ ID NO: 342具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與SEQ ID NO: 640具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與SEQ ID NO: 466具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在一些實施例中，反義寡核苷酸或鄰接核苷酸序列包括10至30個與SEQ ID NO: 566具有至少90%一致性、較佳地100%一致性之核苷酸(長度)或由其組成。 在寡核苷酸長於鄰接核苷酸序列(其與靶核酸互補)之實施例中，表5中之基序序列形成本發明之反義寡核苷酸之鄰接核苷酸序列部分。在一些實施例中，寡核苷酸之序列等效於鄰接核苷酸序列(舉例而言，若不添加生物可裂解連接體)。 應理解，鄰接核鹼基序列(基序序列)可經修飾以(例如)增加核酸酶抗性及/或對靶核酸之結合親和力。修飾闡述於定義及「寡核苷酸設計」部分中。表5列列示每一基序序列之較佳設計。 寡核苷酸設計  寡核苷酸設計係指寡核苷酸序列中之核苷糖修飾模式。本發明寡核苷酸包括糖修飾性核苷且亦可包括DNA或RNA核苷。在一些實施例中，寡核苷酸包括糖修飾性核苷及DNA核苷。將經修飾核苷納入本發明寡核苷酸中可增強寡核苷酸對靶核酸之親和力。在該情形下，經修飾核苷可稱為親和力增強性經修飾核苷酸，經修飾核苷亦可稱為單元。 在一實施例中，寡核苷酸包括至少1個經修飾核苷，例如至少2、至少3、至少4、至少5、至少6、至少7、至少8、至少9、至少10、至少11、至少12、至少13、至少14、至少15或至少16個經修飾核苷。在一實施例中，寡核苷酸包括1至10個經修飾核苷，例如2至8個修飾核苷，例如3至7個經修飾核苷，例如4至6個經修飾核苷，例如3、4、5、6或7個經修飾核苷。 在一實施例中，寡核苷酸包括一或多個糖修飾性核苷，例如2’糖修飾性核苷。較佳地，本發明寡核苷酸包括一或多個獨立地選自由以下組成之群之2’糖修飾性核苷：2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA、2’-胺基-DNA、2’-氟-DNA、阿拉伯糖核酸(ANA)、2’-氟-ANA及LNA核苷。甚至更佳地，一或多個經修飾核苷係鎖核酸(LNA)。 在另一實施例中，寡核苷酸包括至少一個經修飾核苷間鏈接。在一較佳實施例中，鄰接核苷酸序列內之所有核苷間鏈接係硫代磷酸酯或硼烷磷酸酯核苷間鏈接。在一些實施例中，寡核苷酸之鄰接序列中之所有核苷酸間鏈接係硫代磷酸酯鏈接。 在一些實施例中，本發明寡核苷酸包括至少一個LNA核苷，例如1、2、3、4、5、6、7或8個LNA核苷，例如2至6個LNA核苷，例如3至7個LNA核苷，4至6個LNA核苷或3、4、5、6或7個LNA核苷。在一些實施例中，寡核苷酸中之至少75%之經修飾核苷係LNA核苷，舉例而言，80%、例如85%、例如90%之經修飾核苷係LNA核苷。在另一實施例中，寡核苷酸中之所有經修飾核苷皆係LNA核苷。在另一實施例中，寡核苷酸可包括β-D-氧基-LNA及下列LNA核苷中之一或多者：硫代-LNA、胺基-LNA、氧基-LNA及/或ENA (呈β-D或α-L構形或其組合)。在另一實施例中，所有LNA胞嘧啶單元皆係5-甲基-胞嘧啶。在一較佳實施例中，寡核苷酸或鄰接核苷酸序列在核苷酸序列之5’端具有至少1個LNA核苷且在3’端具有至少2個LNA核苷。 在一些實施例中，本發明寡核苷酸包括至少一個係2’-MOE-RNA核苷之經修飾核苷，例如2、3、4、5、6、7、8、9或10個2’-MOE-RNA核苷。在一些實施例中，該等經修飾核苷中之至少一者係2’-氟DNA，例如2、3、4、5、6、7、8、9或10個2’-氟-DNA核苷。 在一些實施例中，本發明寡核苷酸包括至少一個LNA核苷及至少一個2’取代經修飾核苷。 在本發明之一些實施例中，寡核苷酸包括2’糖修飾性核苷及DNA單元。較佳地，寡核苷酸包括LNA及DNA核苷(單元)。較佳地，LNA及DNA單元之組合總數為8-30 (例如10 - 25、較佳地12-22、例如12 - 18、甚至更佳地11-16)。在本發明之一些實施例中，寡核苷酸之核苷酸序列(例如鄰接核苷酸序列)係由至少一個或兩個LNA核苷組成且剩餘核苷係DNA單元。在一些實施例中，寡核苷酸僅包括LNA核苷及天然核苷(例如RNA或DNA、最佳地DNA核苷)，且視情況包括經修飾核苷間鏈接(例如硫代磷酸酯)。 在本發明之一實施例中，本發明寡核苷酸能夠招募RNase H。 本發明寡核苷酸之結構設計可選自間隙聚體、間隙中斷體、頭聚體及尾聚體。 間隙聚體設計  在一較佳實施例中，本發明寡核苷酸具有間隙聚體設計或結構，該間隙聚體設計或結構在本文中亦僅稱為「間隙聚體」。在間隙聚體結構中，寡核苷酸包括至少三個不同結構區域：5’-側翼、間隙及3’-側翼(F-G-F’，以「5 -＞ 3」定向)。在此設計中，在寡核苷酸與靶核酸呈雙螺旋體形式時，側接區F及F’(亦稱為翼區)包括與PD-L1靶核酸互補之經修飾核苷之鄰接序列段，而間隙區G包括能夠招募核酸酶、較佳地內核酸酶(例如RNase，例如RNase H)之核苷酸之鄰接序列段。能夠招募核酸酶、尤其RNase H之核苷可選自由以下組成之群：DNA、α-L-氧基-LNA、2’-氟-ANA及UNA。側接區G之5’及3’端之區域F及F’較佳地包括非核酸酶招募性核苷(具有3’內向結構之核苷)、更佳地一或多個親和力增強性經修飾核苷。在一些實施例中，3’側翼包括至少一個LNA核苷、較佳地至少2個LNA核苷。在一些實施例中，5’側翼包括至少一個LNA核苷。在一些實施例中，5’及3’側接區包括LNA核苷。在一些實施例中，側接區中之所有核苷皆係LNA核苷。在其他實施例中，側接區可包括LNA核苷及其他核苷(混合側翼)，例如DNA核苷及/或非LNA經修飾核苷(例如2’取代核苷)。在此情形下，間隙定義為在5’及3’端側接有由親和力增強性經修飾核苷、較佳地LNA (例如β-D-氧基-LNA)之至少5個RNase H招募性核苷(具有2’內向結構之核苷，較佳係DNA)之鄰接序列。因此，毗鄰間隙區之5’側接區及3’側接區之核苷係經修飾核苷，較佳係非核酸酶招募性核苷。 區域F  連接至區域G之‘5端之區域F(5’側翼或5’翼)包括、含有至少一個經修飾核苷(例如至少2、至少3、至少4、至少5、至少6、至少7個經修飾核苷)或由其組成。在一實施例中，區域F包括1至7個經修飾核苷(例如2至6個經修飾核苷、例如2至5個經修飾核苷、例如2至4個經修飾核苷、例如1至3個經修飾核苷、例如1、2、3或4個經修飾核苷)或由其組成。F區域定義為在該區域之5’端及3’端具有至少一個經修飾核苷。 在一些實施例中，區域F中之經修飾核苷具有3’內向結構。 在一實施例中，區域F中之一或多個經修飾核苷係2’修飾核苷。在一實施例中，區域F中之所有核苷皆係2’修飾核苷。 在另一實施例中，除2’修飾核苷外，區域F亦包括DNA及/或RNA。包括DNA及/或RNA之側翼之特徵在於在F區域之5’端及3’端(毗鄰G區域)具有2’修飾核苷。在一實施例中，區域F包括DNA核苷，例如1至3個鄰接DNA核苷，例如1至3個或1至2個鄰接DNA核苷。側翼中之DNA核苷應較佳地不能招募RNase H。在一些實施例中，F區域中之2’修飾核苷及DNA及/或RNA核苷與1至3個2’修飾核苷及1至3個DNA及/或RNA核苷交替。該等側翼亦可稱為交替側翼。具有交替側翼之寡核苷酸中之5’側翼(區域F)之長度可為4至10個核苷，例如4至8個、例如4至6個核苷，例如4、5、6或7個經修飾核苷。在一些實施例中，僅寡核苷酸之5’側翼係交替性。具有交替核苷之區域F之具體實例如下： 2’1-3 -N’1-4 -2’1-3 2’1-2 -N’1-2 -2’1-2 -N’1-2 -2’1-2 其中2’指示經修飾核苷且N’係RNA或DNA。在一些實施例中，交替側翼中之所有經修飾核苷皆係LNA且N’係DNA。在另一實施例中，區域F中之一或多個2’修飾核苷係選自2’-O-烷基-RNA單元、2’-O-甲基-RNA、2’-胺基-DNA單元、2’-氟-DNA單元、2’-烷氧基-RNA、MOE單元、LNA單元、阿拉伯糖核酸(ANA)單元及2’-氟-ANA單元。 在一些實施例中，F區域包括LNA及2’取代經修飾核苷。該等形式通常稱為混合翼或混合側翼寡核苷酸。 在本發明之一實施例中，區域F中之所有經修飾核苷皆係LNA核苷。在另一實施例中，區域F中之所有核苷皆係LNA核苷。在另一實施例中，區域F中之LNA核苷獨立地選自由以下組成之群：氧基-LNA、硫代-LNA、胺基-LNA、cET及/或ENA (呈β-D或α-L構形或其組合)。在一較佳實施例中，區域F在鄰接序列之5’端至少包括1β-D-氧基LNA單元。 區域G  區域G(間隙區)較佳地包括、含有至少4個(例如至少5、例如至少6、至少7、至少8、至少9、至少10、至少11、至少12、至少13、至少14、至少15或至少16個)能夠招募上文所提及之核酸酶、尤其RNaseH之連續核苷或由其組成。在另一實施例中，區域G包括、含有5至12或6至10或7至9、例如8個能夠招募上文所提及之核酸酶之連續核苷酸單元或由其組成。 區域G中能夠招募核酸酶之核苷單元在一實施例中係選自由以下組成之群：DNA、α-L-LNA、C4’烷基化DNA (如PCT/EP2009/050349及Vester等人，Bioorg. Med. Chem. Lett. 18 (2008) 2296 - 2300 (二者皆以引用方式併入本文中)中所闡述)、阿拉伯糖源核苷(例如ANA及2'F-ANA) (Mangos等人，2003 J. AM. CHEM. SOC. 125, 654-661)、UNA (非鎖定核酸) (如Fluiter等人，Mol. Biosyst., 2009, 10, 1039中所闡述，該文獻以引用方式併入本文中)。UNA係非鎖定核酸，通常其中去除核糖之C2與C3之間之鍵，從而形成非鎖定「糖」殘基。 在另一實施例中，區域G中之至少一個核苷單元係DNA核苷單元，例如1至18個DNA單元、例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或17個DNA單元、較佳地2至17個DNA單元、例如3至16個DNA單元、例如4至15個DNA單元、例如5至14個DNA單元、例如6至13個DNA單元、例如7至12個DNA單元、例如8至11個DNA單元、更佳地8至17個DNA單元或9至16個DNA單元、10至15個DNA單元或11至13個DNA單元、例如8、9、10、11、12、13、14、15、16、17個DNA單元。在一些實施例中，區域G係由100%之DNA單元組成。 在其他實施例中，區域G可由DNA及其他能夠介導RNase H裂解之核苷之混合物組成。區域G可由至少50% DNA、更佳地60%、70%或80% DNA及甚至更佳地90%或95% DNA組成。 在另一實施例中，區域G中之至少一個核苷單元係α-L-LNA核苷單元，例如至少一個α-L-LNA、例如2、3、4、5、6、7、8或9個α-L-LNA。在另一實施例中，區域G包括至少一個係α-L-氧基-LNA之α-L-LNA。在另一實施例中，區域G包括DNA及α-L-LNA核苷單元之組合。 在一些實施例中，區域G中之核苷具有2’內向結構。 在一些實施例中，區域G可包括間隙中斷體核苷，從而產生間隙中斷體寡核苷酸，其能夠招募RNase H。 區域F’  連接至區域G之‘3端之區域F’(3’側翼或3’翼)包括、含有至少一個經修飾核苷(例如至少2、至少3、至少4、至少5、至少6、至少7個經修飾核苷)或由其組成。在一實施例中，區域F’包括1至7個經修飾核苷(例如2至6個經修飾核苷，例如2至4個經修飾核苷，例如1至3個經修飾核苷，例如1、2、3或4個經修飾核苷)或由其組成。F’區域定義為在該區域之5’端及3’端具有至少一個經修飾核苷。 在一些實施例中，區域F’中之經修飾核苷具有3’內向結構。 在一實施例中，區域F’中之一或多個經修飾核苷係2’修飾核苷。在一實施例中，區域F’中之所有核苷皆係2’修飾核苷。 在一實施例中，區域F’中之一或多個經修飾核苷係2’修飾核苷。 在一實施例中，區域F’中之所有核苷皆係2’修飾核苷。在另一實施例中，除2’修飾核苷外，區域F’亦包括DNA或RNA。包括DNA或RNA之側翼之特徵在於在F’區域之5’端(毗鄰G區域)及3’端具有2’修飾核苷。在一實施例中，區域F’包括DNA核苷，例如1至4個鄰接DNA核苷、例如1至3或1至2個鄰接DNA核苷。側翼中之DNA核苷應較佳地不能招募RNase H。在一些實施例中，F’區域中之2’修飾核苷及DNA及/或RNA核苷與1至3個2’修飾核苷及1至3個DNA及/或RNA核苷交替，該等側翼亦可稱為交替側翼。具有交替側翼之寡核苷酸中之3’側翼(區域F’)之長度可為4至10個核苷，例如4至8個、例如4至6個核苷，例如4、5、6或7個經修飾核苷。在一些實施例中，僅寡核苷酸之3’側翼係交替性。具有交替核苷之區域F’之具體實例如下： 2’1-2 -N’1-4 -2’1-4 2’1-2 -N’1-2 -2’1-2 -N’1-2 -2’1-2 其中2’指示經修飾核苷且N’係RNA或DNA。在一些實施例中，交替側翼中之所有經修飾核苷皆係LNA且N’係DNA。在另一實施例中，區域F’中之經修飾核苷係選自2’-O-烷基-RNA單元、2’-O-甲基-RNA、2’-胺基-DNA單元、2’-氟-DNA單元、2’-烷氧基-RNA、MOE單元、LNA單元、阿拉伯糖核酸(ANA)單元及2’-氟-ANA單元。 在一些實施例中，F’區域包括LNA及2’取代經修飾核苷。該等形式通常稱為混合翼或混合側翼寡核苷酸。 在本發明之一實施例中，區域F’中之所有經修飾核苷皆係LNA核苷。在另一實施例中，區域F’中之所有核苷皆係LNA核苷。在另一實施例中，區域F’中之LNA核苷獨立地選自由以下組成之群：氧基-LNA、硫代-LNA、胺基-LNA、cET及/或ENA (呈β-D或α-L構形或其組合)。在一較佳實施例中，區域F’在鄰接序列之3’端至少具有2β-D-氧基LNA單元。 區域D’及D’’  區域D’及D’’可分別連接至區域F之5’端或區域F’之3’端。區域D’或D’’係可選的。 區域D’或D’’可獨立地包括0至5個(例如1至5個，例如2至4個，例如0、1、2、3、4或5個)可與靶核酸互補或非互補之其他核苷酸。就此而言，在一些實施例中，本發明寡核苷酸可包括在5’及/或3’端側接有其他核苷酸能夠調節靶之鄰接核苷酸序列。該等其他核苷酸可用作核酸酶易感性生物可裂解連接體(參見連接體之定義)。在一些實施例中，其他5’及/或3’端核苷與磷酸二酯鏈接連接，且可為DNA或RNA。在另一實施例中，其他5’及/或3’端核苷係可(例如)經包含以增強核酸酶穩定性或便於合成之經修飾核苷。在一實施例中，本發明寡核苷酸在鄰接核苷酸序列之5’或3’端包括區域D’及/或D’’。在另一實施例中，D’及/或D’’區域係由1至5個與靶核酸不互補之磷酸二酯連接之DNA或RNA核苷構成。 本發明之間隙聚體寡核苷酸可由下列各式代表： 5’-F-G-F’-3’；尤其F1-7 -G4-12 -F’1-7 5’-D’-F-G-F’-3’，尤其D’1-3 -F1-7 -G4-12 -F’1-7 5’-F-G-F’-D’’-3’，尤其F1-7 -G4-12 -F’1-7 -D’’1-3 5’-D’-F-G-F’-D’-3’’，尤其D’1-3 -F1-7 -G4-12 -F’1-7 -D’’1-3 區域F、G及F’、D’及D’’中之核苷之較佳數量及類型已闡述於上文中。本發明之寡核苷酸偶聯物具有以共價方式連接至寡核苷酸、尤其上文所呈現之間隙聚體寡核苷酸之5’或3’端之區域C。 在一實施例中，本發明之寡核苷酸偶聯物包括具有式5’-D’-F-G-F’-3’或5’-F-G-F’-D’’-3’之寡核苷酸，其中區域F及F’獨立地包括1 - 7個經修飾核苷，G係6至16個能夠招募RNaseH之核苷之區域且區域D’或D’’包括1 - 5個磷酸二酯連接之核苷。較佳地，區域D’或D’’存在於涵蓋至偶聯物部分之偶聯之寡核苷酸之末端。 具有交替側翼之寡核苷酸之實例可由下列各式代表： 2’1-3 -N’1-4 -2’1-3 -G6-12 -2’1-2 -N’1-4 -2’1-4 2’1-2 -N’1-2 -2’1-2 -N’1-2 -2’1-2 -G6-12 -2’1-2 -N’1-2 -2’1-2 - N’1-2 -2’1-2 F-G6-12 -2’1-2 -N’1-4 -2’1-4 F-G6-12 -2’1-2 -N’1-2 -2’1-2 -N’1-2 -2’1-2 2’1-3 -N’1-4 -2’1-3 -G6-12 -F’ 2’1-2 -N’1-2 -2’1-2 -N1-2 -2’1-2 -G6-12 -F’ 其中側翼由F或F’指示，其僅含有2’修飾核苷(例如LNA核苷)。交替區域及區域F、G及F’、D’及D’’中之核苷之較佳數量及類型已闡述於上文中。 在一些實施例中，寡核苷酸係由16、17、18、19、20、21、22個核苷酸(長度)組成之間隙聚體，其中在與PD-L1靶核酸呈雙螺旋體形式時，區域F及F’中之每一者獨立地由1、2、3或4個與PD-L1靶核酸互補之經修飾核苷單元組成且區域G係由8、9、10、11、12、13、14、15、16、17個能夠招募核酸酶之核苷單元組成，且區域D’係由2磷酸二酯連接之DNA組成。 在另一實施例中，寡核苷酸係間隙聚體，其中區域F及F’中之每一者獨立地由3、4、5或6個經修飾核苷單元(例如含有2’-O-甲氧基乙基-核糖(2’-MOE)之核苷單元或含有2’-氟-去氧核糖之核苷單元及/或LNA單元)組成，且區域G係由8、9、10、11、12、13、14、15、16或17個核苷單元(例如DNA單元或其他核酸酶招募性核苷(例如α-L-LNA)或DNA及核酸酶招募性核苷之混合物)組成。 在另一具體實施例中，寡核苷酸係間隙聚體，其中F及F’區域中之每一者區域係各自由兩個LNA單元組成，且區域G係由12、13、14個核苷單元、較佳地DNA單元組成。此性質之特定間隙聚體設計包含2-12-2、2-13-2及2-14-2。 在另一具體實施例中，寡核苷酸係間隙聚體，其中F及F’中之每一者區域獨立地由三個LNA單元組成，且區域G係由8、9、10、11、12、13或14個核苷單元、較佳地DNA單元組成。具有此性質之特定間隙聚體設計包含3-8-3、3-9-3 3-10-3、3-11-3、3-12-3、3-13-3及3-14-3。 在另一具體實施例中，寡核苷酸係間隙聚體，其中F及F’中之每一者區域各自由4個LNA單元組成，且區域G係由8或9、10、11或12個核苷單元、較佳地DNA單元組成。具有此性質之特定間隙聚體設計包含4-8-4、4-9-4、4-10-4、4-11-4及4-12-4。 具有此性質之特定間隙聚體設計包含選自由6核苷間隙及獨立地翼中之1至4個經修飾核苷組成之群的F-G-F’設計，包含1-6-1、1-6-2、2-6-1、1-6-3、3-6-1、1-6-4、4-6-1、2-6-2、2-6-3、3-6-2、2-6-4、4-6-2、3-6-3、3-6-4及4-6-3間隙聚體。 具有此性質之特定間隙聚體設計包含選自由7核苷間隙及獨立地翼中之1至4個經修飾核苷組成之群的F-G-F’設計，包含1-7-1、2-7-1、1-7-2、1-7-3、3-7-1、1-7-4、4-7-1、2-7-2、2-7-3、3-7-2、2-7-4、4-7-2、3-7-3、3-7-4、4-7-3及4-7-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由8核苷間隙及獨立地翼中之1至4個經修飾核苷組成之群的F-G-F’設計，包含1-8-1、1-8-2、1-8-3、3-8-1、1-8-4、4-8-1、2-8-1、2-8-2、2-8-3、3-8-2、2-8-4、4-8-2、3-8-3、3-8-4、4-8-3及4-8-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由9核苷間隙及獨立地翼中之1至4個經修飾核苷組成之群的F-G-F’設計，包含1-9-1、2-9-1、1-9-2、1-9-3、3-9-1、1-9-4、4-9-1、2-9-2、2-9-3、3-9-2、2-9-4、4-9-2、3-9-3、3-9-4、4-9-3及4-9-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由10核苷間隙組成之群的F-G-F’設計，包含1-10-1、2-10-1、1-10-2、1-10-3、3-10-1、1-10-4、4-10-1、2-10-2、2-10-3、3-10-2、2-10-4、4-10-2、3-10-3、3-10-4、4-10-3及4-10-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由11核苷間隙組成之群的F-G-F’設計，包含1-11-1、2-11-1、1-11-2、1-11-3、3-11-1、1-11-4、4-11-1、2-11-2、2-11-3、3-11-2、2-11-4、4-11-2、3-11-3、3-11-4、4-11-3及4-11-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由12核苷間隙組成之群的F-G-F’設計，包含1-12-1、2-12-1、1-12-2、1-12-3、3-12-1、1-12-4、4-12-1、2-12-2、2-12-3、3-12-2、2-12-4、4-12-2、3-12-3、3-12-4、4-12-3及4-12-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由13核苷間隙組成之群的F-G-F’設計，包含1-13-1、2-13-1、1-13-2、1-13-3、3-13-1、1-13-4、4-13-1、2-13-2、2-13-3、3-13-2、2-13-4、4-13-2、3-13-3、3-13-4、4-13-3及4-13-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由14核苷間隙組成之群的F-G-F’設計，包含1-14-1、2-14-1、1-14-2、1-14-3、3-14-1、1-14-4、4-14-1、2-14-2、2-14-3、3-14-2、2-14-4、4-14-2、3-14-3、3-14-4、4-14-3及4-14-4間隙聚體。 具有此性質之特定間隙聚體設計包含選自由15核苷間隙組成之群的F-G-F’設計，包含1-15-1、2-15-1、1-15-2、1-15-3、3-15-1、1-15-4、4-15-1、2-15-2、2-15-3、3-15-2、2-15-4、4-15-2及3-15-3間隙聚體。 具有此性質之特定間隙聚體設計包含選自由16核苷間隙組成之群的F-G-F’設計，包含1-16-1、2-16-1、1-16-2、1-16-3、3-16-1、1-16-4、4-16-1、2-16-2、2-16-3、3-16-2、2-16-4、4-16-2及3-16-3間隙聚體。 具有此性質之特定間隙聚體設計包含選自由17核苷間隙組成之群的F-G-F’設計，包含1-17-1、2-17-1、1-17-2、1-17-3、3-17-1、1-17-4、4-17-1、2-17-2、2-17-3及3-17-2間隙聚體。 在所有情況下，F-G-F’設計可進一步包含區域D’及/或D’’，該等可具有1、2或3個核苷單元(例如DNA單元，例如2磷酸二酯連接之DNA單元)。較佳地，區域F及F’中之核苷係經修飾核苷，而區域G中之核苷酸較佳係未修飾核苷。 在每一設計中，較佳經修飾核苷係LNA。 在另一實施例中，間隙聚體中之間隙中之所有核苷間鏈接皆係硫代磷酸酯及/或硼烷磷酸酯鏈接。在另一實施例中，間隙聚體中之側翼(F及F’區域)中之所有核苷間鏈接皆係硫代磷酸酯及/或硼烷磷酸酯鏈接。在另一較佳實施例中，間隙聚體中之D’及D’’區域中之所有核苷間鏈接皆係磷酸二酯鏈接。 對於如本文所揭示之特定間隙聚體而言，在胞嘧啶(C)殘基注釋為5-甲基-胞嘧啶時，在各個實施例中，存在於寡核苷酸中之一或多個C可為未修飾C殘基。 在一特定實施例中，間隙聚體係所謂的短聚體，如WO2008/113832 (其以引用方式併入本文中)中所闡述。 其他間隙聚體設計揭示於WO2004/046160、WO2007/146511中且以引用方式併入本文中。 對於本發明之某些實施例而言，寡核苷酸係選自具有CMP-ID-NO: 5_1至743_1及771_1之寡核苷酸化合物之群。 對於本發明之某些實施例而言，寡核苷酸係選自具有以下編號之寡核苷酸化合物之群：CMP-ID-NO 6_1、8_1、9_1、13_1、41_1、42_1、58_1、77_1、92_1、111_1、128_1、151_1、164_1、166_1、169_1、171_1、222_1、233_1、245_1、246_1、250_1、251_1、252_1、256_1、272_1、273_1、287_1、292_1、303_1、314_1、318_1、320_1、324_1、336_1、342_1、343_1、344_1、345_1、346_1、349_1、359_1、360_1、374_1、408_1、409_1、415_1、417_1、424_1、429_1、430_1、458_1、464_1、466_1、474_1、490_1、493_1、512_1、519_1、519_1、529_1、533_1、534_1、547_1、566_1、567_1、578_1、582_1、601_1、619_1、620_1、636_1、637_1、638_1、640_1、645_1、650_1、651_1、652_1、653_1、658_1、659_1、660_1、665_1、678_1、679_1、680_1、682_1、683_1、684_1、687_1、694_1、706_1、716_1、728_1、733_1、734_1及735_1。 在本發明之一較佳實施例中，寡核苷酸係CMP-ID-NO: 287_1。 在本發明之另一較佳實施例中，寡核苷酸係CMP-ID-NO: 342_1。 在本發明之另一較佳實施例中，寡核苷酸係CMP-ID-NO: 640_1。 在本發明之另一較佳實施例中，寡核苷酸係CMP-ID-NO: 466_1。 在本發明之另一較佳實施例中，寡核苷酸係CMP-ID-NO: 566_1。 在本發明之另一實施例中，本發明之寡核苷酸基序及寡核苷酸化合物之鄰接核苷酸序列在鄰接核苷酸序列之5’端包括2至4個其他磷酸二酯連接之核苷(例如區域D’)。在一實施例中，該等核苷用作生物可裂解連接體(參見生物可裂解連接體部分)。在一較佳實施例中，ca (胞苷-腺苷)二核苷酸經由磷酸二酯鏈接連接至鄰接核苷酸序列 (亦即表5中所列示基序序列或寡核苷酸化合物中之任一者)之5’端。在一較佳實施例中，在鄰接核苷酸之其餘部分互補之位置，ca二核苷酸並不與靶序列互補。 在本發明之一些實施例中，寡核苷酸或鄰接核苷酸序列係選自由具有SEQ ID NO: 766、767、768、769及770之核苷酸基序序列組成之群。 在本發明之一些實施例中，寡核苷酸係選自由具有CMP-ID-NO 766_1、767_1、768_1、769_1及770_1之寡核苷酸化合物組成之群。碳水化合物偶聯物部分 碳水化合物偶聯物部分包含(但不限於)半乳糖、乳糖、n-乙醯基半乳糖胺、甘露糖及甘露糖-6-磷酸鹽。可使用碳水化合物偶聯物來增強多種組織(例如肝及/或肌肉)中之遞送或活性。例如參見EP1495769、WO99/65925、Yang等人，Bioconjug Chem (2009) 20(2): 213-21。Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10): 1401-17。 在一些實施例中，碳水化合物偶聯物部分係多價的，舉例而言，2、3或4個相同或不同碳水化合物部分可視情況經由一或多個連接體以共價方式接合至寡核苷酸。在一些實施例中，本發明提供包括本發明寡核苷酸及碳水化合物偶聯物部分之偶聯物。 在一些實施例中，偶聯物部分係或可包括甘露糖或甘露糖-6-磷酸鹽。此尤其可用於靶向肌肉細胞，參見例如US 2012/122801。 能夠結合至去唾液酸醣蛋白受體(ASGPr)之偶聯物部分尤其可用於靶向肝中之肝細胞。在一些實施例中，本發明提供包括本發明寡核苷酸及靶向去唾液酸醣蛋白受體之偶聯物部分之寡核苷酸偶聯物。靶向去唾液酸醣蛋白受體之偶聯物部分包括一或多個能夠以等於或大於半乳糖之親和力結合至去唾液酸醣蛋白受體之碳水化合物部分(結合ASPGr之碳水化合物部分)。許多半乳糖衍生物對去唾液酸醣蛋白受體之親和力已經研究(參見例如：Jobst, S.T.及Drickamer, K. JB.C. 1996, 271, 6686)或輕易使用業內典型方法測定。 本發明之一態樣係包括以下之反義寡核苷酸偶聯物：a)寡核苷酸(區域A)，其包括與PD-L1靶核酸具有至少90%互補性之長度10至30個核苷酸之鄰接核苷酸序列；及b)至少一個靶向去唾液酸醣蛋白受體之偶聯物部分(區域C)，其共價連接至a)中之寡核苷酸。寡核苷酸或鄰接核苷酸序列可如部分「本發明寡核苷酸」、「寡核苷酸設計」及「間隙聚體設計」中之任一者中所述。 在一些實施例中，靶向去唾液酸醣蛋白受體之偶聯物部分包括至少一個選自由以下組成之群之結合ASPGr之碳水化合物部分：半乳糖、半乳糖胺、N-甲醯基-半乳糖胺、N-乙醯基半乳糖胺、N-丙醯基-半乳糖胺、N-正丁醯基-半乳糖胺及N-異丁醯基半乳糖胺。在一些實施例中，靶向去唾液酸醣蛋白受體之偶聯物部分係單價、二價、三價或四價(亦即含有1、2、3或4個能夠結合至去唾液酸醣蛋白受體之末端碳水化合物部分)。較佳地，靶向去唾液酸醣蛋白受體之偶聯物部分係二價，甚至更佳地係三價。在一較佳實施例中，靶向去唾液酸醣蛋白受體之偶聯物部分包括1至3個N-乙醯基半乳糖胺(GalNAc)部分(亦稱為GalNAc偶聯物)。在一些實施例中，寡核苷酸偶聯包括三價N-乙醯基半乳糖胺(GalNAc)部分之靶向去唾液酸醣蛋白受體之偶聯物部分。GalNAc偶聯物已與磷酸二酯、甲基膦酸酯及PNA反義寡核苷酸(例如US 5,994517及Hangeland等人，Bioconjug Chem. 1995 Nov-Dec; 6(6):695-701；Biessen等人，1999 Biochem J. 340, 783-792及Maier等人，2003 Bioconjug Chem 14, 18-29 )及siRNA (例如WO 2009/126933、WO 2012/089352及WO 2012/083046)以及LNA及2'-MOE修飾核苷(WO 2014/076196 WO 2014/207232及WO 2014/179620 (以引用方式併入本文中))一起使用。 為生成靶向去唾液酸醣蛋白受體之偶聯物部分，使ASPGr結合碳水化合物部分(較佳係GalNAc)經由糖之C-l碳連接至支化劑分子。ASPGr結合碳水化合物部分較佳地經由間隔體連接至支化劑分子。較佳間隔體係撓性親水性間隔體(美國專利5885968；Biessen等人。J. Med. Chern. 1995第39卷第1538-1546頁)。較佳撓性親水性間隔體係PEG間隔體。較佳PEG間隔體係PEG3間隔體(三個乙烯單元)。支化劑分子可為任一允許連接兩個或三個結合ASPGr之末端碳水化合物部分且進一步允許將分支點連接至寡核苷酸之小分子。實例性支化劑分子係二離胺酸。二離胺酸分子含有三個胺基團(經由其可連接三個結合ASPGr之碳水化合物部分)及羧基反應性基團(二離胺酸可經由其連接至寡核苷酸)。替代支化劑分子可為雙倍增體或三倍增體，例如由Glen Research所供應者。在一些實施例中，支化劑可選自由以下組成之群：1,3-雙-[5-(4,4'-二甲氧基三苯甲基氧基)戊基醯胺基]丙基-2-[(2-氰基乙基)-(N,N-二異丙基)]亞磷醯胺(Glen Research目錄號：10-1920-xx)、參-2,2,2-[3-(4,4'-二甲氧基三苯甲基氧基)丙基氧基甲基]乙基-[(2-氰基乙基)-(N,N-二異丙基)]-亞磷醯胺(Glen Research目錄號：10-1922-xx)、參-2,2,2-[3-(4,4'-二甲氧基三苯甲基氧基)丙基氧基甲基]亞甲基氧基丙基-[(2-氰基乙基)-(N,N-二異丙基)]-亞磷醯胺及1-[5-(4,4'-二甲氧基-三苯甲基氧基)戊基醯胺基]-3-[5-茀甲氧基-羰基-氧基-戊基醯胺基]-丙基-2-[(2-氰基乙基)-(N,N-二異丙基)]-亞磷醯胺(Glen Research目錄號：10-1925-xx)。WO 2014/179620及PCT申請案第PCT/EP2015/073331號闡述各種GalNAc偶聯物部分之生成(以引用方式併入本文中)。可將一或多個連接體插入支化劑分子與寡核苷酸之間。在一較佳實施例中，連接體係生物可裂解連接體。連接體可選自「連接體」部分及其子部分中所闡述之連接體。 可使用業內已知方法將靶向去唾液酸醣蛋白受體之偶聯物部分、尤其GalNAc偶聯物部分連接至寡核苷酸之3'-端或5'-端。在較佳實施例中，將靶向去唾液酸醣蛋白受體之偶聯物部分連接至寡核苷酸之5’-端。 與siRNA遞送相關之藥物動力學調節劑已闡述於WO2012/083046 (以引用方式併入本文中)中。在一些實施例中，碳水化合物偶聯物部分包括選自由以下組成之群之藥物動力學調節劑：具有16或更多個碳原子之疏水性基團、具有16-20個碳原子之疏水性基團、棕櫚醯基、十六-8-烯醯基、油烯基、(9E,12E)-十八-9,12二烯醯基、二辛醯基及C16-C20醯基及膽固醇。在一較佳實施例中，含有藥物動力學調節劑之碳水化合物偶聯物部分係GalNAc偶聯物。 較佳碳水化合物偶聯物部分包括一至三個結合ASPGr之末端碳水化合物部分、較佳地N-乙醯基半乳糖胺部分。在一些實施例中，碳水化合物偶聯物部分包括三個經由間隔體連接至支化劑分子之結合ASPGr之碳水化合物部分、較佳地N-乙醯基半乳糖胺部分。間隔體分子可長8至30個原子。較佳碳水化合物偶聯物部分包括三個經由PEG間隔體連接至二離胺酸支化劑分子之末端GalNAc部分。較佳地，PEG間隔體係3PEG間隔體。適宜靶向去唾液酸醣蛋白受體之偶聯物部分展示於圖1中。較佳靶向去唾液酸醣蛋白受體之偶聯物部分展示於圖3中。 其他GalNAc偶聯物部分可包含(例如)連接有GalNAc部分之小肽，例如Tyr-Glu-Glu-(胺基己基GalNAc)3 (YEE(ahGalNAc)3；結合至肝細胞上之去唾液酸醣蛋白受體之糖三肽，例如參見Duff等人，Methods Enzymol, 2000, 313, 297)；基於離胺酸之半乳糖簇(例如L3G4；Biessen等人，Cardovasc.  Med., 1999, 214)；及基於膽烷之半乳糖簇(例如用於去唾液酸醣蛋白受體之碳水化合物識別基序)。 在本發明之一些實施例中，反義寡核苷酸偶聯物係選自由以下組成之群：CPM ID NO: 766_2、767_2、768_2、769_2及770_2。 在一較佳實施例中，反義寡核苷酸偶聯物對應於圖4中所表示之化合物。 在另一較佳實施例中，反義寡核苷酸偶聯物對應於圖5中所表示之化合物。 在另一較佳實施例中，反義寡核苷酸偶聯物對應於圖6中所表示之化合物。 在另一較佳實施例中，反義寡核苷酸偶聯物對應於圖7中所表示之化合物。 在另一較佳實施例中，反義寡核苷酸偶聯物對應於圖8中所表示之化合物。連接體 生物可裂解連接體 ( 區域 B) 使用偶聯物通常會增強藥物動力學或藥效動力學性質。然而，偶聯物部分之存在可(例如)經由妨礙雜交或核酸酶招募(例如RNAseH)之立體阻礙來干擾寡核苷酸針對其預期靶之活性。在寡核苷酸(區域A或第一區域)與偶聯物部分(區域C或第三區域)之間使用生理上不穩定鍵(生物可裂解連接體)使得因存在偶聯物部分而改良性質，同時確保在靶組織處偶聯基團不妨礙寡核苷酸之有效活性。 在含有生理上不穩定鍵之分子到達適當細胞內及/或細胞外環境時，該不穩定鍵自發發生裂解。舉例而言，在分子進入經酸化胞內體時，pH不穩定鍵可發生裂解。因此，pH不穩定鍵可視為胞內體可裂解鍵。酶可裂解鍵可在暴露於酶(例如存在於胞內體或溶酶體中或細胞質中者)時發生裂解。二硫鍵可在分子進入細胞質之較高還原環境時發生裂解。因此，二硫化物可視為細胞質可裂解鍵。如本文中所使用，pH不穩定鍵係在酸性條件(pH＜7)下選擇性斷裂之不穩定鍵。該等鍵亦可稱為胞內體不穩定鍵，此乃因細胞胞內體及溶酶體之pH小於7。 對於與用於靶向遞送之偶聯物部分締合之生物可裂解連接體而言，較佳地，靶組織(例如肌肉、肝、腎或腫瘤)中所看到之裂解速率大於發現於血清中者。用於測定靶組織與血清中之裂解或S1核酸酶裂解之程度(%)之適宜方法闡述於「材料及方法」部分中。在一些實施例中，本發明偶聯物中之生物可裂解連接體(亦稱為生理上不穩定連接體或核酸酶易感連接體或區域B)與標準相比至少約20%裂解，例如至少約30%裂解、例如至少約40%裂解、例如至少約50%裂解、例如至少約60%裂解、例如至少約70%裂解、例如至少約75%裂解。 在一些實施例中，本發明之寡核苷酸偶聯物包括三個區域：i)第一區域(區域A)，其包括10 - 25個與靶核酸互補之鄰接核苷酸；ii)第二區域(區域B)，其包括生物可裂解連接體；及iii)第三區域(區域C)，其包括偶聯物部分，例如靶向去唾液酸醣蛋白受體之偶聯物部分，其中第三區域共價連接至以共價方式連接至第一區域之第二區域。 在本發明之一實施例中，寡核苷酸偶聯物在鄰接核苷酸序列(區域A)與靶向去唾液酸醣蛋白受體之偶聯物部分(區域C)之間包括生物可裂解連接體(區域B)。 在一些實施例中，生物可裂解連接體可位於與靶核酸互補之鄰接核苷酸(區域A)之5’端及/或3’端。在一較佳實施例中，生物可裂解連接體位於5’端。 在一些實施例中，可裂解連接體易感可(例如)表現於靶細胞中之核酸酶。在一些實施例中，生物可裂解連接體係由2至5個連續磷酸二酯鏈接構成。連接體可為短區域(例如1 - 10，如連接體定義中所詳述)磷酸二酯連接之核苷。在一些實施例中，生物可裂解連接體區B中之核苷(視情況獨立地)選自由以下組成之群：DNA及RNA或其不干擾核酸酶裂解之修飾。不干擾核酸酶裂解之DNA及RNA核苷修飾可為非天然核鹼基。某些糖修飾性核苷亦可容許核酸酶裂解，例如α-L-氧基-LNA。在一些實施例中，區域B中之所有核苷包括(視情況獨立地) 2’-OH核糖糖(RNA)或2’-H糖-亦即RNA或DNA。在一較佳實施例中，區域B之至少兩個連續核苷係DNA或RNA核苷(例如至少3或4或5個連續DNA或RNA核苷)。在一甚至更佳實施例中，區域B之核苷係DNA核苷。較佳地，區域B係由1至5或1至4 (例如2、3、4)個連續磷酸二酯連接之DNA核苷組成。在較佳實施例中，區域B較短以便其不招募RNaseH。在一些實施例中，區域B包括不超過3或不超過4個連續磷酸二酯連接之DNA及/或RNA核苷(例如DNA核苷)。 在區域B係由磷酸二酯連接之核苷構成之情形下，區域A及B可一起形成連接至區域C之寡核苷酸。在此背景下，區域A與區域B之不同之處可在於，區域A始於至少一個、較佳地至少兩個對靶核酸具有增加之結合親和力之經修飾核苷(例如LNA或具有2’取代糖部分之核苷)且區域A自身能夠調節靶核酸在相關細胞系中之表現。另外，若區域A包括DNA或RNA核苷，則該等核苷與核酸酶抗性核苷間鏈接(例如硫代磷酸酯或硼烷磷酸酯)連接。另一方面，區域B在DNA與RNA核苷之間包括磷酸二酯鏈接。在一些實施例中，區域B並不與靶核酸互補或相對於靶核酸包括至少50%失配。 在一些實施例中，區域B並不與靶核酸序列或與區域A中之靶核酸互補之鄰接核苷酸互補。 在一些實施例中，區域B與靶核酸序列互補。就此而言，區域A及B一起可形成與靶序列互補之單一鄰接序列。 在本發明之一些態樣中，第一區域(區域A)及第二區域(區域B)之間之核苷間鏈接可視為第二區域之一部分。 在一些實施例中，基於存在於靶組織或細胞或子細胞腔室中之主要內核酸酶裂解酶，選擇區域B中之鹼基序列以提供最佳內核酸酶裂解位點。就此而言，藉由自靶組織及非靶組織分離細胞提取物，可基於期望靶細胞(例如肝/肝細胞)中與非靶細胞(例如腎)相比之優先裂解活性來選擇用於區域B中之內核酸酶裂解序列。就此而言，可針對期望組織/細胞最佳化化合物關於靶下調之功效。 在一些實施例中，區域B包括序列AA、AT、AC、AG、TA、TT、TC、TG、CA、CT、CC、CG、GA、GT、GC或GG之二核苷酸，其中C可為5-甲基胞嘧啶，及/或T可經U代替。較佳地，核苷間鏈接係磷酸二酯鏈接。在一些實施例中，區域B包括序列AAA、AAT、AAC、AAG、ATA、ATT、ATC、ATG、ACA、ACT、ACC、ACG、AGA、AGT、AGC、AGG、TAA、TAT、TAC、TAG、TTA、TTT、TTC、TAG、TCA、TCT、TCC、TCG、TGA、TGT、TGC、TGG、CAA、CAT、CAC、CAG、CTA、CTG、CTC、CTT、CCA、CCT、CCC、CCG、CGA、CGT、CGC、CGG、GAA、GAT、GAC、CAG、GTA、GTT、GTC、GTG、GCA、GCT、GCC、GCG、GGA、GGT、GGC及GGG之三核苷酸，其中C可為5-甲基胞嘧啶及/或T可經U代替。較佳地，核苷間鏈接係磷酸二酯鏈接。在一些實施例中，區域B包括序列AAAX、AATX、AACX、AAGX、ATAX、ATTX、ATCX、ATGX、ACAX、ACTX、ACCX、ACGX、AGAX、AGTX、AGCX、AGGX、TAAX、TATX、TACX、TAGX、TTAX、TTTX、TTCX、TAGX、TCAX、TCTX、TCCX、TCGX、TGAX、TGTX、TGCX、TGGX、CAAX、CATX、CACX、CAGX、CTAX、CTGX、CTCX、CTTX、CCAX、CCTX、CCCX、CCGX、CGAX、CGTX、CGCX、CGGX、GAAX、GATX、GACX、CAGX、GTAX、GTTX、GTCX、GTGX、GCAX、GCTX、GCCX、GCGX、GGAX、GGTX、GGCX及GGGX之三核苷酸，其中X可選自由以下組成之群：A、T、U、G、C及其類似物，其中C可為5-甲基胞嘧啶及/或T可經U代替。較佳地，核苷間鏈接係磷酸二酯鏈接。應識別到，在提及(天然)核鹼基A、T、U、G、C時，該等核鹼基可經用作等效天然核鹼基之核鹼基類似物(例如具有互補核苷之鹼基對)取代。其他連接體 ( 區域 Y) 連接體可具有至少兩個官能基，一個官能基用於連接至寡核苷酸且另一個用於連接至偶聯物部分。實例性連接體官能基可為親電性以用於與寡核苷酸或偶聯物部分上之親核性基團進行反應，或為親核性以用於與親電性基團進行反應。在一些實施例中，連接體官能基包含胺基、羥基、羧酸、硫醇、胺基磷酸酯、硫代磷酸酯、磷酸酯、亞磷酸酯、不飽和基團(例如雙鍵或三鍵)及諸如此類。一些實例性連接體(區域Y)包含8-胺基-3,6-二氧雜辛酸(ADO)、4-(N-馬來醯亞胺基甲基)環己烷-l-甲酸琥珀醯亞胺基酯(SMCC)、6-胺基己酸(AHEX或AHA)、6-胺基己基氧基、4-胺基丁酸、4-胺基環己基甲酸、琥珀醯亞胺基4-(N-馬來醯亞胺基甲基)環己烷- l-羧基-(6-醯胺基-己酸酯) (LCSMCC)、間馬來醯亞胺基-苯甲酸琥珀醯亞胺基酯(MBS)、N-e-馬來醯亞胺基-辛酸琥珀醯亞胺基酯(EMCS)、6-(β-馬來醯亞胺基-丙醯胺基)己酸琥珀醯亞胺基酯(SMPH)、琥珀醯亞胺基N-(a-馬來醯亞胺基乙酸酯) (AMAS)、4-(p-馬來醯亞胺基苯基)丁酸琥珀醯亞胺基酯(SMPB)、β -丙胺酸(β-ALA)、苯基甘胺酸(PHG)、4-胺基環己酸(ACHC)、β -(環丙基)丙胺酸(β-CYPR)、胺基十二烷酸(ADC)、伸烷基二醇、聚乙二醇、胺基酸及諸如此類。在一些實施例中，連接體(區域Y)係胺基烷基，例如C2 - C36胺基烷基(包含(例如) C6 - C12胺基烷基)。在一較佳實施例中，連接體(區域Y)係C6胺基烷基。可(例如)使用(例如經保護)胺基烷基胺基磷酸胺基酯烷基酯添加至寡核苷酸(區域A或區域A-B)中以作為標準寡核苷酸合成之一部分。胺基烷基與寡核苷酸之間之鏈接基團可(例如)為硫代磷酸酯或磷酸二酯或本文所提及其他核苷鏈接基團中之一者。胺基烷基以共價方式連接至寡核苷酸之5’或3’端。市售胺基烷基連接體係(例如) 3'-胺基-修飾劑試劑(用於寡核苷酸之3’端處之鏈接)，且對於寡核苷酸之5 '端處之鏈接而言，可使用5'-胺基-修飾劑C6。該等試劑可自Glen Research Corporation (Sterling, Va.)獲得。該等化合物或類似物在Krieg等人，Antisense Research and Development 1991, 1, 161中用於將螢光黃連接至寡核苷酸之5'-末端。業內已知眾多種其他連接體基團且可用於將偶聯物部分連接至寡核苷酸。許多有用連接體基團之綜述可參見(例如) Antisense Research and Applications, S. T. Crooke及B. Lebleu編輯，CRC Press, Boca Raton, Fla., 1993，第303-350頁。其他化合物(例如吖啶)經由聚亞甲基鏈接連接至寡核苷酸之3'-末端磷酸酯基團(Asseline等人，Proc. Natl. Acad. Sci. USA 1984, 81, 3297)。任一上述基團皆可用作單一連接體(區域Y)或與一或多個其他連接體組合使用(區域Y-Y’或區域Y-B或B-Y)。 連接體及其製備寡核苷酸偶聯物之用途已在業內廣泛提供，例如WO 96/11205及WO 98/52614及U.S. Pat. No. 4,948,882；5,525,465；5,541,313；5,545,730；5,552,538；5,580,731；5,486,603；5,608,046；4,587,044；4,667,025；5,254,469；5,245,022；5,112,963；5,391,723；5,510475；5,512,667；5,574,142；5,684,142；5,770,716；6,096,875；6,335,432；及6,335,437；WO 2012/083046，該等案件中之每一者之全部內容皆以引用方式併入本文中。 製造方法  在另一態樣中，本發明提供製造本發明寡核苷酸之方法，其包括使核苷酸單元進行反應且由此形成包括於寡核苷酸中之以共價方式連接之鄰接核苷酸單元。較佳地，該方法使用亞磷醯胺化學(例如參見Caruthers等人，1987, Methods in Enzymology第154卷，第287-313頁)。在另一實施例中，該方法進一步包括使鄰接核苷酸序列與偶聯物部分(配體)進行反應。在另一態樣中，提供製造本發明組合物之方法，其包括混合本發明之寡核苷酸或偶聯寡核苷酸與醫藥上可接受之稀釋劑、溶劑、載劑、鹽及/或佐劑。 醫藥組合物  在另一態樣中，本發明提供醫藥組合物，其包括任一上文所提及之寡核苷酸及/或寡核苷酸偶聯物及醫藥上可接受之稀釋劑、溶劑、載劑、鹽及/或佐劑。醫藥上可接受之稀釋劑包含磷酸緩衝鹽水(PBS)且醫藥上可接受之鹽包含(但不限於)鈉鹽及鉀鹽。在一些實施例中，醫藥上可接受之稀釋劑係無菌磷酸鹽緩衝鹽水。在一些實施例中，寡核苷酸以50 - 300µM溶液之濃度用於醫藥上可接受之稀釋劑中。 用於本發明中之適宜調配物可參見Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.，第17版，1985。關於藥物遞送方法之簡要綜述，參見(例如) Langer (Science 249:1527-1533, 1990)。WO2007/031091提供醫藥上可接受之稀釋劑、載劑及佐劑之其他適宜及較佳實例(以引用方式併入本文中)。適宜劑量、調配物、投與途徑、組合物、劑型、與其他治療劑之組合、前藥調配物亦提供於WO2007/031091中。 可混合本發明之寡核苷酸或寡核苷酸偶聯物與醫藥上可接受之活性或惰性物質以用於製備醫藥組合物或調配物。組合物及調配醫藥組合物之方法取決於諸多準則，包含(但不限於)投與途徑、疾病程度或擬投與劑量。 該等組合物可藉由習用滅菌技術進行滅菌，或可無菌過濾。可將所得水溶液包裝以供按原樣使用或將其凍乾，將凍乾製劑在投與之前與無菌水性載劑組合。製劑之pH通常介於3與11之間，更佳地介於5與9之間或介於6與8之間，且最佳地介於7與8之間(例如7至7.5)。可將呈固體形式之所得組合物包裝成多個單一劑量單元，每一單元含有固定量之上文所提及之一或多種藥劑，例如呈錠劑或膠囊之密封包裝形式。亦可將呈固體形式之組合物包裝成用於撓性量之容器，例如呈設計用於局部施加之乳霜或軟膏之可擠壓管形式。 在一些實施例中，本發明之寡核苷酸或寡核苷酸偶聯物係前藥。特定而言，對於寡核苷酸偶聯物而言，在將前藥遞送至作用位點(例如靶細胞)後，偶聯物部分立即與寡核苷酸分離。 應用  本發明之寡核苷酸或寡核苷酸偶聯物可用作用於(例如)診斷、治療及預防之研究試劑。 在研究中，可使用該等寡核苷酸或寡核苷酸偶聯物來特異性調節PD-L1蛋白在細胞(例如活體外細胞培養物)及實驗動物中之合成，由此促進靶之功能分析或其作為用於治療干預之靶之有用性的評價。通常，藉由降解或抑制產生蛋白質之mRNA (由此防止形成蛋白質)或藉由降解或抑制產生蛋白質之基因或mRNA之調節劑來達成靶調節。 若在研究或診斷中採用本發明寡核苷酸，則靶核酸可為cDNA或衍生自DNA或RNA之合成核酸。 本發明提供調節表現PD-L1之靶細胞中之PD-L1表現之活體內或活體外方法，該方法包括向該細胞投與有效量之本發明之寡核苷酸或寡核苷酸偶聯物。 在一些實施例中，靶細胞係哺乳動物細胞、尤其人類細胞。靶細胞可為哺乳動物中之組織之活體外細胞培養物或活體內細胞形成部分。在較佳實施例中，靶細胞存在於肝中。肝靶細胞可選自實質細胞(例如肝細胞)及非實質細胞(例如庫弗氏細胞、LSEC、星形細胞(或Ito細胞)、膽管上皮細胞及肝相關白血球(包含T細胞及NK細胞))。在一些實施例中，靶細胞係抗原呈遞細胞。抗原呈遞細胞在其表面上顯示與種類I或種類II主要組織相容性複合物(MHC)複合之外來抗原。在一些實施例中，抗原呈遞細胞表現種類II MHC (亦即職業性抗原呈遞細胞，例如樹突狀細胞、巨噬球及B細胞)。 在診斷中，可使用寡核苷酸藉由北方印漬(北方印漬)、原位雜交或類似技術來檢測及量化細胞及組織中之PD-L1表現。 對於治療而言，可將本發明之寡核苷酸或寡核苷酸偶聯物或其醫藥組合物投與懷疑患有疾病或病症之動物或人類，該疾病或病症可藉由降低PD-L1表現、尤其藉由降低肝靶細胞中之PD-L1表現來予以緩解或治療。 本發明提供治療或預防疾病之方法，其包括向患有或易感該疾病之個體投與治療或預防有效量之本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物。 本發明亦係關於用作藥劑之本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物。 本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物通常係以有效量來投與。 本發明亦提供本發明之所闡述寡核苷酸或寡核苷酸偶聯物或醫藥組合物之用途，其用以製造用於治療如本文所提及之疾病或病症之藥劑。在一實施例中，該疾病係選自a)病毒性肝感染，例如HBV、HCV及HDV；b)寄生蟲感染，例如瘧疾、弓蟲症、利什曼病及錐蟲病；及c)肝癌或肝中轉移。 在一實施例中，本發明係關於用於治療選自病毒或寄生蟲感染之疾病或病症之寡核苷酸、寡核苷酸偶聯物或醫藥組合物。在另一實施例中，該疾病係選自a)病毒性肝感染，例如HBV、HCV及HDV；b)寄生蟲感染，例如瘧疾、弓蟲症、利什曼病及錐蟲病；及c)肝癌或肝中轉移。 如本文所提及之疾病或病症與免疫耗竭有關。特定而言，疾病或病症與病毒特異性T細胞反應之耗竭有關。在一些實施例中，可藉由降低PD-L1表現來緩解或治療疾病或病症。 本發明方法較佳用於治療或預防與免疫耗竭有關之疾病。 在本發明之一實施例中，本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物可用於恢復針對肝癌或肝中轉移之免疫反應。 在本發明之一實施例中，本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物可用於恢復針對病原體之免疫反應。在一些實施例中，病原體可發現於肝中。病原體可為病毒或寄生蟲，尤其係本文所闡述者。在一較佳實施例中，病原體係HBV。 本發明另外係關於如本文所定義之寡核苷酸、寡核苷酸偶聯物或醫藥組合物之用途，其用以製造用於恢復針對如本文所提及病毒或寄生蟲感染之免疫性之藥劑。 本發明之寡核苷酸或寡核苷酸偶聯物或醫藥組合物可用於治療病毒感染、尤其影響PD-1路徑之肝中之病毒感染(例如參見Kapoor及Kottilil 2014 Future Virol第9卷第565-585頁及Salem及El-Badawy 2015 World J Hepatol第7卷第2449-2458頁)。病毒性肝感染可選自由以下組成之群：肝炎病毒、尤其HBV、HCV及HDV、尤其該等感染之慢性形式。在一實施例中，使用本發明之寡核苷酸或寡核苷酸偶聯物或醫藥組合物來治療HBV、尤其慢性HBV。慢性HBV感染之指標係循環中之高病毒負荷值(HBV DNA)及甚至較高含量之空HBsAg顆粒(＞100倍過量病毒體)。 本發明之寡核苷酸或寡核苷酸偶聯物亦可用於治療與HIV共感染發生之病毒性肝感染。可使用本發明之寡核苷酸或寡核苷酸偶聯物或醫藥組合物治療之其他病毒感染係lcmv (淋巴球性脈絡叢腦膜炎病毒)及HIV (呈單菌性感染形式)、HSV-1及HSV-2及其他皰疹病毒。該等病毒並非感染肝細胞傾向性，然而，其可對PDL1下調敏感。 在一些實施例中，免疫性或免疫反應之恢復涉及改良T細胞及/或NK細胞反應及/或緩解T細胞耗竭，特定而言，恢復HBV特異性T細胞反應、HCV特異性T細胞反應及或HDV特異性T細胞反應。可(例如)將T細胞反應之改良評價為與對照(例如在治療之前之值或在媒劑治療個體中之值)相比肝中之T細胞增加、尤其CD8+及/或CD4+ T細胞增加。在另一實施例中，病毒特異性CD8+ T細胞與對照相比有所恢復或增加，特定而言，HBV特異性CD8+ T細胞或HCV特異性CD8+ T細胞或HDV特異性CD8+ T細胞與對照相比有所恢復或增加。在一較佳實施例中，在使用本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物治療之個體中，與對照相比，HBV s抗原(HBsAg)特異性CD8+ T細胞及/或HBV e抗原(HBeAg)特異性CD8+ T細胞及/或HBV核心抗原(HBcAg)特異性CD8+ T細胞有所增加。較佳地，HBV抗原特異性CD8+ T細胞產生一或多種細胞介素(例如干擾素-γ(IFN-γ)或腫瘤壞死因子α (TNF-α))。尤其在肝中觀察到上述CD8+ T細胞之增加。在與對照相比時，本文所闡述之增加應在統計學上顯著。較佳地，在與對照相比時，增加至少20% (例如25%，例如50%，例如75%)。在另一實施例中，藉由本發明之寡核苷酸或寡核苷酸偶聯物活化天然殺手(NK)細胞及/或天然殺手T (NKT)細胞。 本發明之寡核苷酸或寡核苷酸偶聯物或醫藥組合物可用於治療寄生蟲感染、尤其影響PD-1路徑之寄生蟲感染(例如參見Bhadra等人， 2012 J Infect Dis第206卷第125-134頁；Bhadra等人， 2011 Proc Natl Acad Sci U S A第108卷第9196-9201頁；Esch等人， J Immunol第191卷第5542-5550頁；Freeman及Sharpe 2012 Nat Immunol第13卷第113-115頁；Gutierrez等人， 2011 Infect Immun第79卷第1873-1881頁；Joshi等人， 2009 PLoS Pathog第5卷e1000431；Liang等人， 2006 Eur J Immunol第36卷第58-64頁；Wykes等人， 2014 Front Microbiol第5卷第249頁)。寄生蟲感染可選自由以下組成之群：瘧疾、弓蟲症、利什曼病及錐蟲病。瘧疾感染係由瘧原蟲屬、尤其物種間日瘧原蟲、三日瘧原蟲及惡性瘧原蟲之原生動物引起。 弓蟲症係由弓蟲(Toxoplasma gondii )引起之寄生蟲疾病。利什曼病係由利什曼原蟲(Leishmania )屬之原生動物寄生蟲引起之疾病。 錐蟲病係藉由布魯氏錐蟲(Trypanosoma )屬之原生動物引起。查加斯病(Chaga disease)係由物種克魯斯錐蟲(Trypanosoma cruzi )引起之熱帶形式，且睡眠疾病係由物種布魯氏錐蟲(Trypanosoma brucei )引起。 在一些實施例中，免疫性恢復涉及恢復寄生蟲特異性T細胞及NK細胞反應、尤其瘧原蟲屬特異性T細胞反應、弓蟲特異性T細胞及NK細胞反應、利什曼原蟲特異性T細胞及NK細胞反應、克魯斯錐蟲特異性T細胞及NK細胞反應或布魯氏錐蟲特異性T細胞及NK細胞反應。在另一實施例中，恢復寄生蟲特異性CD8+ T細胞及NK細胞反應。 投與  可局部(例如皮膚、吸入、眼部或耳部)或經腸(例如經口或經由胃腸道)或非經腸(例如靜脈內、皮下、肌內、大腦內、大腦心室內或鞘內)投與本發明之寡核苷酸或醫藥組合物。 在一較佳實施例中，藉由非經腸途徑(包含靜脈內、動脈內、皮下、腹膜腔內或肌內注射或輸注、鞘內或顱內(例如大腦內)或心室內、玻璃體內投與)投與本發明之寡核苷酸或醫藥組合物。在一實施例中，經靜脈內投與活性寡核苷酸或寡核苷酸偶聯物。在另一實施例中，經皮下投與活性寡核苷酸或寡核苷酸偶聯物。 在一些實施例中，以0.1 - 15 mg/kg (例如0.1 - 10 mg/kg，例如0.2 - 10 mg/kg，例如0.25 - 10 mg/kg，例如0.1 - 5 mg/kg，例如0.2 - 5 mg/kg，例如0.25 - 5 mg/kg)之劑量投與本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物。可每週一次、每第2週，每第三週或甚至每月一次進行投與。 組合療法  在一些實施例中，將本發明之寡核苷酸、寡核苷酸偶聯物或醫藥組合物與另一治療劑用於組合治療。治療劑可為(例如)用於上述疾病或病症之標準護理。 為治療慢性HBV感染，推薦使用抗病毒藥物及免疫系統調節劑之組合作為標準護理。有效針對HBV之抗病毒藥物係(例如)核苷(酸)類似物。5種核苷(酸)類似物已經許可用於治療HBV，亦即拉米夫定(lamivudine) (Epivir)、阿德福韋(adefovir) (Hepsera)、替諾福韋(tenofovir) (Viread)、替比夫定(telbivudine) (Tyzeka)、恩替卡韋(entecavir) (Baraclude)，該等藥劑有效阻抑病毒複製(HBV DNA)，但對HBsAg含量並無效應。其他抗病毒藥物包含利巴韋林(ribavirin)及HBV抗體療法(單株或多株)。免疫系統調節劑可為(例如)干擾素α-2a及聚乙二醇化干擾素α-2a (Pegasys)或TLR7激動劑(例如GS-9620)或治療疫苗。IFN-α治療僅展示降低病毒負荷之極輕微效應，但產生一定程度之HBsAg下降，但該下降極低(在48週療法之後＜10%)。 本發明之寡核苷酸或寡核苷酸偶聯物亦可與其他有效抵抗HBV之抗病毒藥物(例如闡述於WO2012/145697及WO 2014/179629中之反義寡核苷酸或闡述於WO 2005/014806、WO 2012/024170、WO 2012/2055362、WO 2013/003520及WO 2013/159109中之siRNA分子)進行組合。 在組合療法中投與本發明之寡核苷酸或寡核苷酸偶聯物與其他藥劑時，可依序或同時將其投與個體。或者，本發明之醫藥組合物可包括本發明之寡核苷酸或寡核苷酸偶聯物以及如本文所闡述醫藥上可接受之賦形劑及業內已知之另一治療性或預防性藥劑的組合。 實施例  本發明之下列實施例可與本文所闡述之任一其他實施例組合使用。 1. 一種反義寡核苷酸，其包括長度為10至30個核苷酸之鄰接核苷酸序列或由其組成且能夠降低PD-L1表現。 2. 如實施例1之寡核苷酸，其中該鄰接核苷酸序列與PD-L1靶核酸至少90%互補。 3. 如實施例1或2之寡核苷酸，其中該鄰接核苷酸序列與選自由以下組成之群之靶核酸互補：SEQ ID NO: 1、SEQ ID NO: 2及/或SEQ ID NO: 3。 4. 如實施例1至3之寡核苷酸，其中該鄰接核苷酸序列與SEQ ID NO: 1上之位置1及15720內之區域互補。 5. 如實施例1至4之寡核苷酸，其中該寡核苷酸能夠以低於-10 kcal之ΔG°雜交至選自由以下組成之群之靶核酸：SEQ ID NO: 1、SEQ ID NO: 2及/或SEQ ID NO: 3。 6. 如實施例1至5之寡核苷酸，其中該鄰接核苷酸序列與該靶核酸之子序列互補，其中該子序列係選自由以下組成之群：SEQ ID NO: 1上之位置371至3068、5467至12107、15317至15720、15317-18083、15317至19511及18881至19494。 7. 如實施例6之寡核苷酸，其中該子序列係選自由以下組成之群：SEQ ID NO: 1上之位置7300至7333、8028至8072、9812至9859、11787至11873及15690至15735。 8. 如實施例2至7之寡核苷酸，其中該靶核酸係RNA。 9. 如實施例8之寡核苷酸，其中該RNA係mRNA。 10. 如實施例9之寡核苷酸，其中該mRNA係mRNA前體或成熟mRNA。 11. 如實施例1至10之寡核苷酸，其中該鄰接核苷酸序列包括至少14個鄰接核苷酸、尤其15、16、17、18、19、20、21、22、23或24個鄰接核苷酸或由其組成。 12. 如實施例1至10之寡核苷酸，其中該鄰接核苷酸序列包括16至20個核苷酸或由其組成。 13. 如實施例1至10之寡核苷酸，其中該寡核苷酸包括14至35個核苷酸(長度)或由其組成。 14. 如實施例13之寡核苷酸，其中該寡核苷酸包括18至22個核苷酸(長度)或由其組成。 15. 如實施例1至14之寡核苷酸，其中該寡核苷酸或鄰接核苷酸序列係單鏈。 16. 如實施例1至15之寡核苷酸，其中該鄰接核苷酸序列與該靶核酸之子序列互補，其中該子序列係選自由以下組成之群：A7、A26、A43、A119、A142、A159、A160、A163、A169、A178、A179、A180、A189、A201、A202、A204、A214、A221、A224、A226、A243、A254、A258、269、A274、A350、A360、A364、A365、A370、A372、A381、A383、A386、A389、A400、A427、A435及A438。 17. 如實施例16之寡核苷酸，其中該子序列係選自由以下組成之群：A221、A360、A180、A160及A269。 18. 如實施例1至17之寡核苷酸，其中該寡核苷酸並非siRNA且並不自我互補。 19. 如實施例1至18之寡核苷酸，其中該鄰接核苷酸序列包括選自SEQ ID NO: 5至743或771之序列或由其組成。 20. 如實施例1至19之寡核苷酸，其中該鄰接核苷酸序列包括選自以下之序列或由其組成：SEQ ID NO: 6、8、9、13、41、42、58、77、92、111、128、151、164、166、169、171、222、233、245、246、250、251、252、256、272、273、287、292、303、314、318、320、324、336、342、343、344、345、346、349、359、360、374、408、409、415、417、424、429、430、458、464、466、474、490、493、512、519、519、529、533、534、547、566、567、578、582、601、619、620、636、637、638、640、645、650、651、652、653、658、659、660、665、678、679、680、682、683、684、687、694、706、716、728、733、734及735。 21. 如實施例1至20之寡核苷酸，其中該鄰接核苷酸序列包括選自SEQ ID NO:466、640、342、287及566之序列或由其組成。 22. 如實施例1至21之寡核苷酸，其中該鄰接核苷酸序列與其互補靶核酸相比具有零至三個失配。 23. 如實施例22之寡核苷酸，其中該鄰接核苷酸序列與該靶核酸相比具有一個失配。 24. 如實施例22之寡核苷酸，其中該鄰接核苷酸序列與該靶核酸相比具有兩個失配。 25. 如實施例22之寡核苷酸，其中該鄰接核苷酸序列與該靶核酸序列完全互補。 26. 如實施例1至25之寡核苷酸，其包括一或多個經修飾核苷。 27. 如實施例26之寡核苷酸，其中該一或多個經修飾核苷係高親和力經修飾核苷。 28. 如實施例26或27之寡核苷酸，其中該一或多個經修飾核苷係2’糖修飾性核苷。 29. 如實施例28之寡核苷酸，其中該一或多個2’糖修飾性核苷獨立地選自由以下組成之群：2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA、2’-胺基-DNA、2’-氟-DNA、2’-氟-ANA及LNA核苷。 30. 如實施例28之寡核苷酸，其中該一或多個經修飾核苷係LNA核苷。 31. 如實施例30之寡核苷酸，其中該經修飾LNA核苷係氧基-LNA。 32. 如實施例31之寡核苷酸，其中該經修飾核苷係β-D-氧基-LNA。 33. 如實施例30之寡核苷酸，其中該經修飾核苷係硫代-LNA。 34. 如實施例30之寡核苷酸，其中該經修飾核苷係胺基-LNA。 35. 如實施例30之寡核苷酸，其中該經修飾核苷係cET。 36. 如實施例30之寡核苷酸，其中該經修飾核苷係ENA。 37. 如實施例30之寡核苷酸，其中該經修飾LNA核苷係選自β-D-氧基-LNA、α-L-氧基-LNA、β-D-胺基-LNA、α-L-胺基-LNA、β-D-硫代-LNA、α-L-硫代-LNA、(S)cET、(R)cETβ-D-ENA及α-L-ENA。 38. 如實施例30至37之寡核苷酸，其中除該經修飾LNA核苷外存在至少一個2’取代經修飾核苷。 39. 如實施例38之寡核苷酸，其中該2’取代經修飾核苷係選自由以下組成之群：2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA (MOE)、2’-胺基-DNA、2’-氟-DNA、2’-氟-ANA。 40. 如實施例1至39中任一項之寡核苷酸，其中該寡核苷酸包括至少一個經修飾核苷間鏈接。 41. 如實施例40之寡核苷酸，其中該經修飾核苷間鏈接係核酸酶抗性。 42. 如實施例40或41之寡核苷酸，其中該鄰接核苷酸序列內之至少50%之該等核苷間鏈接係硫代磷酸酯核苷間鏈接或硼烷磷酸酯核苷間鏈接。 43. 如實施例40或41之寡核苷酸，其中該鄰接核苷酸序列內之所有該等核苷間鏈接皆係硫代磷酸酯核苷間鏈接。 44. 如實施例1至43之寡核苷酸，其中該寡核苷酸能夠招募RNase H。 45. 如實施例44之寡核苷酸，其中該寡核苷酸係間隙聚體。 46. 如實施例44或45之寡核苷酸，其中該寡核苷酸係式5’-F-G-F’-3’之間隙聚體，其中區域F及F’獨立地包括1至7個經修飾核苷或由其組成且G係能夠招募RNaseH之6至16個核苷之區域。 47. 如實施例44或45之寡核苷酸，其中該間隙聚體具有式5’-D’-F-G-F’-3’或5’-F-G-F’-D’’-3’，其中區域F及F’獨立地包括1至7個經修飾核苷，G係能夠招募RNaseH之6至16個核苷之區域且區域D’或D’’包括1至5個磷酸二酯連接之核苷。 48. 如實施例47之寡核苷酸，其中D’或D’’係可選的。 49. 如實施例47之寡核苷酸，其中區域D’係由兩個磷酸二酯連接之核苷組成。 50. 如實施例49之寡核苷酸，其中該等磷酸二酯連接之核苷係ca (胞苷-腺苷)。 51. 如實施例46或47之寡核苷酸，其中該經修飾核苷係獨立地選自由以下組成之群之2’糖修飾性核苷：2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA、2’-胺基-DNA、2’-氟-DNA、阿拉伯糖核酸(ANA)、2’-氟-ANA及LNA核苷。 52. 如實施例46至51之寡核苷酸，其中區域F及F’中之該等經修飾核苷中之一或多者係LNA核苷。 53. 如實施例52之寡核苷酸，其中區域F及F’中之所有該等經修飾核苷皆係LNA核苷。 54. 如實施例53之寡核苷酸，其中區域F及F’係由LNA核苷組成。 55. 如實施例52至54之寡核苷酸，其中區域F及F’中之所有該等經修飾核苷皆係氧基-LNA核苷。 56. 如實施例52之寡核苷酸，其中區域F或F’中之至少一者進一步包括至少一個獨立地選自由以下組成之群之2’取代經修飾核苷：2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA、2’-胺基-DNA及2’-氟-DNA。 57. 如實施例46至56之寡核苷酸，其中區域G中之該等招募RNaseH之核苷獨立地選自DNA、α-L-LNA、C4’烷基化DNA、ANA及2'F-ANA及UNA。 58. 如實施例57之寡核苷酸，其中區域G中之該等核苷係DNA及/或α-L-LNA核苷。 59. 如實施例57或58之寡核苷酸，其中區域G由至少75%之DNA核苷組成。 60. 如實施例1至59之寡核苷酸，其中該寡核苷酸係選自CMP ID NO: 5_1至743_1及771_1 (表5)中之任一者。 61. 如實施例1至60之寡核苷酸，其中該寡核苷酸係選自由以下組成之群：CMP ID NO: 6_1、8_1、9_1、13_1、41_1、42_1、58_1、77_1、92_1、111_1、128_1、151_1、164_1、166_1、169_1、171_1、222_1、233_1、245_1、246_1、250_1、251_1、252_1、256_1、272_1、273_1、287_1、292_1、303_1、314_1、318_1、320_1、324_1、336_1、342_1、343_1、344_1、345_1、346_1、349_1、359_1、360_1、374_1、408_1、409_1、415_1、417_1、424_1、429_1、430_1、458_1、464_1、466_1、474_1、490_1、493_1、512_1、519_1、519_1、529_1、533_1、534_1、547_1、566_1、567_1、578_1、582_1、601_1、619_1、620_1、636_1、637_1、638_1、640_1、645_1、650_1、651_1、652_1、653_1、658_1、659_1、660_1、665_1、678_1、679_1、680_1、682_1、683_1、684_1、687_1、694_1、706_1、716_1、728_1、733_1、734_1及735_1。 62. 如實施例1至61之寡核苷酸，其中該寡核苷酸係選自由以下組成之群：CMP ID NO:287_1、342_1、466_1、640_1、566_1、766_1、767_1、768_1、769_1及770_1。 63. 一種反義寡核苷酸偶聯物，其包括 a. 如實施例1至62中任一項之寡核苷酸(區域A)；及 b. 至少一個以共價方式連接至該寡核苷酸之偶聯物部分(區域C)。 64. 如實施例63之寡核苷酸偶聯物，其中該偶聯物部分係選自碳水化合物、細胞表面受體配體、藥物物質、激素、親脂性物質、聚合物、蛋白質、肽、毒素、維他命、病毒蛋白質或其組合。 65. 如實施例63或64之寡核苷酸偶聯物，其中該偶聯物部分係含有碳水化合物之部分。 66. 如實施例65之寡核苷酸偶聯物，其中該碳水化合物偶聯物部分包括至少一個共價連接至如實施例1至62中任一項之寡核苷酸之靶向去唾液酸醣蛋白受體之部分。 67. 如實施例66之寡核苷酸偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分包括至少一個選自由以下組成之群之碳水化合物部分：半乳糖、半乳糖胺、N-甲醯基-半乳糖胺、N-乙醯基半乳糖胺、N-丙醯基-半乳糖胺、N-正丁醯基-半乳糖胺及N-異丁醯基半乳糖胺。 68. 如實施例66或67之寡核苷酸偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分係單價、二價、三價或四價。 69. 如實施例68之寡聚物偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分係由二至四個末端GalNAc部分、將每一GalNAc部分連接至支化劑分子之PEG間隔體組成。 70. 如實施例66至69之寡核苷酸偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分係三價N-乙醯基半乳糖胺(GalNAc)部分。 71. 如實施例66至70之寡核苷酸偶聯物，其中該偶聯物部分係選自圖1中之三價GalNAc部分中之一者。 72. 如實施例71之寡核苷酸偶聯物，其中該偶聯物部分係圖3中之三價GalNAc部分。 73. 如實施例63至72之寡核苷酸偶聯物，其中連接體存在於該寡核苷酸或鄰接寡核苷酸序列與該偶聯物部分之間。 74. 如實施例73之寡核苷酸偶聯物，其中該連接體係生理上不穩定連接體(區域B)。 75. 如實施例74之寡核苷酸偶聯物，其中該生理上不穩定連接體係核酸酶易感連接體。 76. 如實施例74或75之寡核苷酸偶聯物，其中該生理上不穩定連接體係由2至5個連續磷酸二酯鏈接構成。 77. 如實施例76之寡核苷酸偶聯物，其中該生理上不穩定連接體等效於實施例47至50中所呈現之區域D’或D’’。 78. 如實施例63至77中任一項之寡核苷酸偶聯物，其中該寡核苷酸偶聯物係選自CMP ID NO: 766_2、767_2、768_2、769_2及770_2。 79. 如實施例78之寡核苷酸偶聯物，其中該寡核苷酸偶聯物係選自圖4、5、6、7及8中所表示之寡核苷酸偶聯物。 80. 如實施例63至76之寡核苷酸偶聯物，其與未偶聯寡核苷酸相比顯示靶細胞中之PD-L1之改良抑制，或在肝與脾之間顯示改良之細胞分佈，或顯示偶聯寡核苷酸在肝中之改良之細胞攝取。 81. 一種醫藥組合物，其包括如實施例1至62之寡核苷酸或如實施例63至80之偶聯物及醫藥上可接受之稀釋劑、載劑、鹽及/或佐劑。 82. 一種製造如實施例1至62之寡核苷酸之方法，其包括使核苷酸單元進行反應，由此形成包括於該寡核苷酸中之以共價方式連接之鄰接核苷酸單元。 83. 如實施例82之方法，其進一步包括使該鄰接核苷酸序列與非核苷酸偶聯物部分進行反應。 84. 一種製造如實施例81之組合物之方法，其包括混合該寡核苷酸與醫藥上可接受之稀釋劑、載劑、鹽及/或佐劑。 85. 一種調節表現PD-L1之靶細胞中之PD-L1表現之活體內或活體外方法，該方法包括向該細胞投與有效量之如實施例1至62之寡核苷酸或如實施例63-80之偶聯物或如實施例81之醫藥組合物。 86. 一種治療或預防疾病之方法，其包括向患有或易患該疾病之個體投與治療或預防有效量之如實施例1至62之寡核苷酸或如實施例63-80之偶聯物或如實施例81之醫藥組合物。 87. 一種恢復針對病毒或寄生蟲之免疫性之方法，其包括向感染病毒或寄生蟲之個體投與治療或預防有效量之如實施例63至80之寡核苷酸偶聯物或如實施例1至62之寡核苷酸或如實施例81之醫藥組合物。 88. 如實施例87之方法，免疫性之該恢復係在與對照相比時增加肝中之對一或多種HBV抗原具有特異性之CD8+ T細胞。 89. 如實施例1至62之寡核苷酸或如實施例63至80之偶聯物或如實施例81之醫藥組合物，其用作用於治療或預防個體之疾病之藥劑。 90. 一種如實施例1至62之寡核苷酸或如實施例63至80之偶聯物之用途，其用以製備用於治療或預防個體之疾病之藥劑。 91. 如實施例1至62之寡核苷酸或如實施例63至80之偶聯物或如實施例81之醫藥組合物，其用於恢復針對病毒或寄生蟲之免疫性。 92. 如實施例91之用途，其中免疫性之該恢復係在與對照相比時增加肝中之對一或多種HBV抗原具有特異性之CD8+ T細胞。 93. 如實施例92之用途，其中該HBV抗原係HBsAg。 94. 如實施例86至93之方法、寡核苷酸或用途，其中該疾病與PD-L1之活體內活性有關。 95. 如實施例86至94之方法、寡核苷酸或用途，其中該疾病與抗原呈遞細胞中之增加之PD-L1表現有關。 96. 如實施例95之方法、寡核苷酸或用途，其中與未治療或在使用如實施例1至62之寡核苷酸或如實施例63至80之偶聯物或如實施例81之醫藥組合物進行治療之前之表現相比，該PD-L1降低至少30%或至少40%或至少50%或至少60%或至少70%或至少80%或至少90%或至少95%。 97. 如實施例86至95之方法、寡核苷酸或用途，其中該疾病係選自病毒性肝感染或寄生蟲感染。 98. 如實施例98之方法、寡核苷酸或用途，其中該病毒感染係HBV、HCV或HDV。 99. 如實施例86至95之方法、寡核苷酸或用途，其中該疾病係慢性HBV。 100. 如實施例98之方法、寡核苷酸或用途，其中該寄生蟲感染係瘧疾、弓蟲症、利什曼病或錐蟲病。 101. 如實施例86至100之方法、寡核苷酸或用途，其中該個體係哺乳動物。 102. 如實施例101之方法、寡核苷酸或用途，其中該哺乳動物係人類。 實例  材料及方法基序序列及寡核苷酸化合物 表5：靶向人類PD-L1轉錄物(SEQ ID NO: 1)之寡核苷酸基序序列(由SEQ ID NO指示)、該等序列之設計以及基於基序序列設計之特定反義寡核苷酸化合物(由CMP ID NO指示)之列表。 SEQ ID NO 基序序列 設計 寡核苷酸化合物 CMP ID NO ID NO: 1 上之起點 dG 5 taattggctctactgc 2-11-3 TAattggctctacTGC 5_1 236 -20 6 tcgcataagaatgact 4-10-2 TCGCataagaatgaCT 6_1 371 -19 7 tgaacacacagtcgca 2-12-2 TGaacacacagtcgCA 7_1 382 -19 8 ctgaacacacagtcgc 3-10-3 CTGaacacacagtCGC 8_1 383 -22 9 tctgaacacacagtcg 3-11-2 TCTgaacacacagtCG 9_1 384 -19 10 ttctgaacacacagtc 3-11-2 TTCtgaacacacagTC 10_1 385 -17 11 acaagtcatgttacta 2-11-3 ACaagtcatgttaCTA 11_1 463 -16 12 acacaagtcatgttac 2-12-2 ACacaagtcatgttAC 12_1 465 -14 13 cttacttagatgctgc 2-11-3 CTtacttagatgcTGC 13_1 495 -20 14 acttacttagatgctg 2-11-3 ACttacttagatgCTG 14_1 496 -18 15 gacttacttagatgct 3-11-2 GACttacttagatgCT 15_1 497 -19 16 agacttacttagatgc 2-11-3 AGacttacttagaTGC 16_1 498 -18 17 gcaggaagagacttac 3-10-3 GCAggaagagactTAC 17_1 506 -20 18 aataaattccgttcagg 4-9-4 AATAaattccgttCAGG 18_1 541 -22 19 gcaaataaattccgtt 3-10-3 GCAaataaattccGTT 19_2 545 -18 19 gcaaataaattccgtt 4-8-4 GCAAataaattcCGTT 19_1 545 -20 20 agcaaataaattccgt 4-9-3 AGCAaataaattcCGT 20_1 546 -20 21 cagagcaaataaattcc 4-10-3 CAGAgcaaataaatTCC 21_1 548 -21 22 tggacagagcaaataaat 4-11-3 TGGAcagagcaaataAAT 22_1 551 -19 23 atggacagagcaaata 4-8-4 ATGGacagagcaAATA 23_1 554 -20 24 cagaatggacagagca 2-11-3 CAgaatggacagaGCA 24_1 558 -21 25 ttctcagaatggacag 3-11-2 TTCtcagaatggacAG 25_1 562 -17 26 ctgaactttgacatag 4-8-4 CTGAactttgacATAG 26_1 663 -20 27 aagacaaacccagactga 2-13-3 AAgacaaacccagacTGA 27_1 675 -21 28 tataagacaaacccagac 4-10-4 TATAagacaaacccAGAC 28_1 678 -22 29 ttataagacaaacccaga 4-10-4 TTATaagacaaaccCAGA 29_1 679 -23 30 tgttataagacaaaccc 4-10-3 TGTTataagacaaaCCC 30_1 682 -22 31 tagaacaatggtacttt 4-9-4 TAGAacaatggtaCTTT 31_1 708 -20 32 gtagaacaatggtact 4-10-2 GTAGaacaatggtaCT 32_1 710 -19 33 aggtagaacaatggta 3-10-3 AGGtagaacaatgGTA 33_1 712 -19 34 aagaggtagaacaatgg 4-9-4 AAGAggtagaacaATGG 34_1 714 -21 35 gcatccacagtaaatt 2-12-2 GCatccacagtaaaTT 35_1 749 -17 36 gaaggttatttaattc 2-11-3 GAaggttatttaaTTC 36_1 773 -13 37 ctaatcgaatgcagca 4-9-3 CTAAtcgaatgcaGCA 37_1 805 -22 38 tacccaatctaatcga 3-10-3 TACccaatctaatCGA 38_1 813 -20 39 tagttacccaatctaa 3-10-3 TAGttacccaatcTAA 39_1 817 -19 40 catttagttacccaat 3-10-3 CATttagttacccAAT 40_1 821 -18 41 tcatttagttacccaa 3-10-3 TCAtttagttaccCAA 41_1 822 -19 42 ttcatttagttaccca 2-10-4 TTcatttagttaCCCA 42_1 823 -22 43 gaattaatttcatttagt 4-10-4 GAATtaatttcattTAGT 43_1 829 -19 44 cagtgaggaattaattt 4-9-4 CAGTgaggaattaATTT 44_1 837 -20 45 ccaacagtgaggaatt 4-8-4 CCAAcagtgaggAATT 45_1 842 -21 46 cccaacagtgaggaat 3-10-3 CCCaacagtgaggAAT 46_1 843 -22 47 tatacccaacagtgagg 2-12-3 TAtacccaacagtgAGG 47_1 846 -21 48 ttatacccaacagtgag 2-11-4 TTatacccaacagTGAG 48_1 847 -21 49 tttatacccaacagtga 3-11-3 TTTatacccaacagTGA 49_1 848 -21 50 cctttatacccaacag 3-10-3 CCTttatacccaaCAG 50_1 851 -23 51 taacctttatacccaa 4-8-4 TAACctttatacCCAA 51_1 854 -22 52 aataacctttataccca 3-10-4 AATaacctttataCCCA 52_1 855 -23 53 gtaaataacctttata 3-11-2 GTAaataacctttaTA 53_1 859 -14 54 actgtaaataacctttat 4-10-4 ACTGtaaataacctTTAT 54_1 860 -20 55 atatatatgcaatgag 3-11-2 ATAtatatgcaatgAG 55_1 903 -14 56 agatatatatgcaatg 2-12-2 AGatatatatgcaaTG 56_1 905 -12 57 gagatatatatgcaat 3-10-3 GAGatatatatgcAAT 57_1 906 -15 58 ccagagatatatatgc 2-11-3 CCagagatatataTGC 58_1 909 -19 59 caatattccagagatat 4-9-4 CAATattccagagATAT 59_1 915 -20 60 gcaatattccagagata 4-10-3 GCAAtattccagagATA 60_1 916 -22 61 agcaatattccagagat 3-11-3 AGCaatattccagaGAT 61_1 917 -22 62 cagcaatattccagag 3-9-4 CAGcaatattccAGAG 62_1 919 -22 63 aatcagcaatattccag 4-9-4 AATCagcaatattCCAG 63_1 921 -23 64 acaatcagcaatattcc 4-9-4 ACAAtcagcaataTTCC 64_1 923 -21 65 actaagtagttacacttct 2-14-3 ACtaagtagttacactTCT 65_1 957 -20 66 ctaagtagttacacttc 4-11-2 CTAAgtagttacactTC 66_1 958 -18 67 gactaagtagttacactt 3-12-3 GACtaagtagttacaCTT 67_1 959 -20 68 tgactaagtagttaca 3-9-4 TGActaagtagtTACA 68_1 962 -19 69 ctttgactaagtagtta 4-10-3 CTTTgactaagtagTTA 69_1 964 -19 70 ctctttgactaagtag 3-10-3 CTCtttgactaagTAG 70_1 967 -19 71 gctctttgactaagta 4-10-2 GCTCtttgactaagTA 71_1 968 -21 72 ccttaaatactgttgac 2-11-4 CCttaaatactgtTGAC 72_1 1060 -20 73 cttaaatactgttgac 2-12-2 CTtaaatactgttgAC 73_1 1060 -13 74 tccttaaatactgttg 3-10-3 TCCttaaatactgTTG 74_1 1062 -18 75 tctccttaaatactgtt 4-11-2 TCTCcttaaatactgTT 75_1 1063 -19 76 tatcatagttctcctt 2-10-4 TAtcatagttctCCTT 76_1 1073 -21 77 agtatcatagttctcc 3-10-3 AGTatcatagttcTCC 77_1 1075 -22 78 gagtatcatagttctc 2-11-3 GAgtatcatagttCTC 78_1 1076 -18 79 agagtatcatagttct 2-10-4 AGagtatcatagTTCT 79_1 1077 -18 79 agagtatcatagttct 3-10-3 AGAgtatcatagtTCT 79_2 1077 -19 80 cagagtatcatagttc 3-10-3 CAGagtatcatagTTC 80_1 1078 -18 81 ttcagagtatcatagt 4-10-2 TTCAgagtatcataGT 81_1 1080 -18 82 cttcagagtatcatag 3-9-4 CTTcagagtatcATAG 82_1 1081 -19 83 ttcttcagagtatcata 4-11-2 TTCTtcagagtatcaTA 83_1 1082 -19 84 tttcttcagagtatcat 3-10-4 TTTcttcagagtaTCAT 84_1 1083 -20 85 gagaaaggctaagttt 4-9-3 GAGAaaggctaagTTT 85_1 1099 -19 86 gacactcttgtacatt 2-10-4 GAcactcttgtaCATT 86_1 1213 -19 87 tgagacactcttgtaca 2-13-2 TGagacactcttgtaCA 87_1 1215 -18 88 tgagacactcttgtac 2-11-3 TGagacactcttgTAC 88_1 1216 -18 89 ctttattaaactccat 2-10-4 CTttattaaactCCAT 89_1 1266 -18 90 accaaactttattaaa 4-10-2 ACCAaactttattaAA 90_1 1272 -14 91 aaacctctactaagtg 4-10-2 AAACctctactaagTG 91_1 1288 -16 92 agattaagacagttga 2-11-3 AGattaagacagtTGA 92_1 1310 -16 93 aagtaggagcaagaggc 2-12-3 AAgtaggagcaagaGGC 93_1 1475 -22 94 aaagtaggagcaagagg 4-10-3 AAAGtaggagcaagAGG 94_1 1476 -20 95 gttaagcagccaggag 2-12-2 GTtaagcagccaggAG 95_1 1806 -20 96 agggtaggatgggtag 2-12-2 AGggtaggatgggtAG 96_1 1842 -20 97 aagggtaggatgggta 3-11-2 AAGggtaggatgggTA 97_1 1843 -20 98 caagggtaggatgggt 2-12-2 CAagggtaggatggGT 98_2 1844 -20 98 caagggtaggatgggt 3-11-2 CAAgggtaggatggGT 98_1 1844 -21 99 ccaagggtaggatggg 2-12-2 CCaagggtaggatgGG 99_1 1845 -22 100 tccaagggtaggatgg 2-12-2 TCcaagggtaggatGG 100_1 1846 -20 101 cttccaagggtaggat 4-10-2 CTTCcaagggtaggAT 101_1 1848 -21 102 atcttccaagggtagga 3-12-2 ATCttccaagggtagGA 102_1 1849 -22 103 agaagtgatggctcatt 2-11-4 AGaagtgatggctCATT 103_1 1936 -21 104 aagaagtgatggctcat 3-10-4 AAGaagtgatggcTCAT 104_1 1937 -21 105 gaagaagtgatggctca 3-11-3 GAAgaagtgatggcTCA 105_1 1938 -21 106 atgaaatgtaaactggg 4-9-4 ATGAaatgtaaacTGGG 106_1 1955 -21 107 caatgaaatgtaaactgg 4-10-4 CAATgaaatgtaaaCTGG 107_1 1956 -20 108 gcaatgaaatgtaaactg 4-10-4 GCAAtgaaatgtaaACTG 108_1 1957 -20 109 agcaatgaaatgtaaact 4-10-4 AGCAatgaaatgtaAACT 109_1 1958 -20 110 gagcaatgaaatgtaaac 4-10-4 GAGCaatgaaatgtAAAC 110_1 1959 -19 111 tgaattcccatatccga 2-12-3 TGaattcccatatcCGA 111_1 1992 -22 112 agaattatgaccatat 2-11-3 AGaattatgaccaTAT 112_1 2010 -15 113 aggtaagaattatgacc 3-10-4 AGGtaagaattatGACC 113_1 2014 -21 114 tcaggtaagaattatgac 4-10-4 TCAGgtaagaattaTGAC 114_1 2015 -22 115 cttcaggtaagaattatg 4-10-4 CTTCaggtaagaatTATG 115_1 2017 -21 116 tcttcaggtaagaatta 4-9-4 TCTTcaggtaagaATTA 116_1 2019 -20 117 cttcttcaggtaagaat 4-9-4 CTTCttcaggtaaGAAT 117_1 2021 -21 118 tcttcttcaggtaagaa 4-10-3 TCTTcttcaggtaaGAA 118_1 2022 -20 119 tcttcttcaggtaaga 3-10-3 TCTtcttcaggtaAGA 119_1 2023 -20 120 tggtctaagagaagaag 3-10-4 TGGtctaagagaaGAAG 120_1 2046 -20 121 gttggtctaagagaag 4-9-3 GTTGgtctaagagAAG 121_1 2049 -19 123 cagttggtctaagagaa 2-11-4 CAgttggtctaagAGAA 123_1 2050 -20 124 gcagttggtctaagagaa 3-13-2 GCAgttggtctaagagAA 124_1 2050 -22 122 agttggtctaagagaa 3-9-4 AGTtggtctaagAGAA 122_1 2050 -20 126 gcagttggtctaagaga 2-13-2 GCagttggtctaagaGA 126_1 2051 -21 125 cagttggtctaagaga 4-10-2 CAGTtggtctaagaGA 125_1 2051 -21 127 gcagttggtctaagag 2-11-3 GCagttggtctaaGAG 127_1 2052 -21 128 ctcatatcagggcagt 2-10-4 CTcatatcagggCAGT 128_1 2063 -24 129 cacacatgttctttaac 4-11-2 CACAcatgttctttaAC 129_1 2087 -18 130 taaatacacacatgttct 3-11-4 TAAatacacacatgTTCT 130_1 2092 -19 131 gtaaatacacacatgttc 4-11-3 GTAAatacacacatgTTC 131_1 2093 -19 132 tgtaaatacacacatgtt 4-10-4 TGTAaatacacacaTGTT 132_1 2094 -22 133 gatcatgtaaatacacac 4-10-4 GATCatgtaaatacACAC 133_1 2099 -20 134 agatcatgtaaatacaca 4-10-4 AGATcatgtaaataCACA 134_1 2100 -21 135 caaagatcatgtaaatacac 4-12-4 CAAAgatcatgtaaatACAC 135_1 2101 -19 136 acaaagatcatgtaaataca 4-12-4 ACAAagatcatgtaaaTACA 136_1 2102 -20 137 gaatacaaagatcatgta 4-10-4 GAATacaaagatcaTGTA 137_1 2108 -20 138 agaatacaaagatcatgt 4-10-4 AGAAtacaaagatcATGT 138_1 2109 -20 139 cagaatacaaagatcatg 4-10-4 CAGAatacaaagatCATG 139_1 2110 -21 140 gcagaatacaaagatca 4-9-4 GCAGaatacaaagATCA 140_1 2112 -22 141 aggcagaatacaaagat 4-11-2 AGGCagaatacaaagAT 141_1 2114 -19 142 aaggcagaatacaaaga 4-10-3 AAGGcagaatacaaAGA 142_1 2115 -19 143 attagtgagggacgaa 3-10-3 ATTagtgagggacGAA 143_1 2132 -18 144 cattagtgagggacga 2-11-3 CAttagtgagggaCGA 144_1 2133 -20 145 gagggtgatggattag 2-11-3 GAgggtgatggatTAG 145_1 2218 -19 146 ttaggagtaataaagg 2-10-4 TTaggagtaataAAGG 146_1 2241 -14 147 ttaatgaatttggttg 3-11-2 TTAatgaatttggtTG 147_1 2263 -13 148 ctttaatgaatttggt 2-12-2 CTttaatgaatttgGT 148_1 2265 -14 149 catggattacaactaa 4-10-2 CATGgattacaactAA 149_1 2322 -16 150 tcatggattacaacta 2-11-3 TCatggattacaaCTA 150_1 2323 -16 151 gtcatggattacaact 3-11-2 GTCatggattacaaCT 151_1 2324 -18 152 cattaaatctagtcat 2-10-4 CAttaaatctagTCAT 152_1 2335 -16 153 gacattaaatctagtca 4-10-3 GACAttaaatctagTCA 153_1 2336 -19 154 agggacattaaatcta 4-10-2 AGGGacattaaatcTA 154_1 2340 -18 155 caaagcattataacca 4-9-3 CAAAgcattataaCCA 155_1 2372 -18 156 acttactaggcagaag 2-10-4 ACttactaggcaGAAG 156_1 2415 -19 157 cagagttaactgtaca 4-10-2 CAGAgttaactgtaCA 157_1 2545 -20 158 ccagagttaactgtac 4-10-2 CCAGagttaactgtAC 158_1 2546 -20 159 gccagagttaactgta 2-12-2 GCcagagttaactgTA 159_1 2547 -20 160 tgggccagagttaact 2-12-2 TGggccagagttaaCT 160_1 2550 -21 161 cagcatctatcagact 2-12-2 CAgcatctatcagaCT 161_1 2576 -19 162 tgaaataacatgagtcat 3-11-4 TGAaataacatgagTCAT 162_1 2711 -19 163 gtgaaataacatgagtc 3-10-4 GTGaaataacatgAGTC 163_1 2713 -19 164 tctgtttatgtcactg 4-10-2 TCTGtttatgtcacTG 164_1 2781 -20 165 gtctgtttatgtcact 4-10-2 GTCTgtttatgtcaCT 165_1 2782 -22 166 tggtctgtttatgtca 2-10-4 TGgtctgtttatGTCA 166_1 2784 -21 167 ttggtctgtttatgtc 4-10-2 TTGGtctgtttatgTC 167_1 2785 -20 168 tcacccattgtttaaa 2-12-2 TCacccattgtttaAA 168_1 2842 -15 169 ttcagcaaatattcgt 2-10-4 TTcagcaaatatTCGT 169_1 2995 -17 170 gtgtgttcagcaaatat 3-10-4 GTGtgttcagcaaATAT 170_1 2999 -21 171 tctattgttaggtatc 3-10-3 TCTattgttaggtATC 171_1 3053 -18 172 attgcccatcttactg 2-12-2 ATtgcccatcttacTG 172_1 3118 -19 173 tattgcccatcttact 3-11-2 TATtgcccatcttaCT 173_1 3119 -21 174 aaatattgcccatctt 2-11-3 AAatattgcccatCTT 174_1 3122 -17 175 ataaccttatcataca 3-11-2 ATAaccttatcataCA 175_1 3174 -16 176 tataaccttatcatac 2-11-3 TAtaaccttatcaTAC 176_1 3175 -14 177 ttataaccttatcata 3-11-2 TTAtaaccttatcaTA 177_1 3176 -14 178 tttataaccttatcat 3-10-3 TTTataaccttatCAT 178_1 3177 -16 179 actgctattgctatct 2-11-3 ACtgctattgctaTCT 179_1 3375 -19 180 aggactgctattgcta 2-11-3 AGgactgctattgCTA 180_1 3378 -21 181 gaggactgctattgct 3-11-2 GAGgactgctattgCT 181_1 3379 -22 182 acgtagaataataaca 2-12-2 ACgtagaataataaCA 182_1 3561 -11 183 ccaagtgatataatgg 2-10-4 CCaagtgatataATGG 183_1 3613 -19 184 ttagcagaccaagtga 2-10-4 TTagcagaccaaGTGA 184_1 3621 -21 185 gtttagcagaccaagt 2-12-2 GTttagcagaccaaGT 185_1 3623 -19 186 tgacagtgattatatt 2-12-2 TGacagtgattataTT 186_1 3856 -13 187 tgtccaagatattgac 4-10-2 TGTCcaagatattgAC 187_1 3868 -18 188 gaatatcctagattgt 3-10-3 GAAtatcctagatTGT 188_1 4066 -18 189 caaactgagaatatcc 2-11-3 CAaactgagaataTCC 189_1 4074 -16 190 gcaaactgagaatatc 3-11-2 GCAaactgagaataTC 190_1 4075 -16 191 tcctattacaatcgta 3-11-2 TCCtattacaatcgTA 191_1 4214 -19 192 ttcctattacaatcgt 4-10-2 TTCCtattacaatcGT 192_1 4215 -19 193 actaatgggaggattt 2-12-2 ACtaatgggaggatTT 193_1 4256 -15 194 tagttcagagaataag 2-12-2 TAgttcagagaataAG 194_1 4429 -13 195 taacatatagttcaga 2-11-3 TAacatatagttcAGA 195_1 4436 -15 196 ataacatatagttcag 3-11-2 ATAacatatagttcAG 196_1 4437 -14 197 cataacatatagttca 2-12-2 CAtaacatatagttCA 197_1 4438 -13 198 tcataacatatagttc 2-12-2 TCataacatatagtTC 198_1 4439 -12 199 tagctcctaacaatca 4-10-2 TAGCtcctaacaatCA 199_1 4507 -22 200 ctccaatctttgtata 4-10-2 CTCCaatctttgtaTA 200_1 4602 -20 201 tctccaatctttgtat 4-10-2 TCTCcaatctttgtAT 201_1 4603 -19 202 tctatttcagccaatc 2-12-2 TCtatttcagccaaTC 202_1 4708 -17 203 cggaagtcagagtgaa 3-10-3 CGGaagtcagagtGAA 203_1 4782 -19 204 ttaagcatgaggaata 4-10-2 TTAAgcatgaggaaTA 204_1 4798 -16 205 tgattgagcacctctt 3-10-3 TGAttgagcacctCTT 205_1 4831 -22 206 gactaattatttcgtt 3-11-2 GACtaattatttcgTT 206_1 4857 -15 207 tgactaattatttcgt 3-10-3 TGActaattatttCGT 207_1 4858 -17 208 gtgactaattatttcg 3-10-3 GTGactaattattTCG 208_1 4859 -17 209 ctgcttgaaatgtgac 4-10-2 CTGCttgaaatgtgAC 209_1 4870 -20 210 cctgcttgaaatgtga 2-11-3 CCtgcttgaaatgTGA 210_1 4871 -21 211 atcctgcttgaaatgt 2-10-4 ATcctgcttgaaATGT 211_1 4873 -20 212 attataaatctattct 3-10-3 ATTataaatctatTCT 212_1 5027 -13 213 gctaaatactttcatc 2-11-3 GCtaaatactttcATC 213_1 5151 -16 214 cattgtaacataccta 2-10-4 CAttgtaacataCCTA 214_1 5251 -19 215 gcattgtaacatacct 2-12-2 GCattgtaacatacCT 215_1 5252 -18 216 taatattgcaccaaat 2-12-2 TAatattgcaccaaAT 216_1 5295 -13 217 gataatattgcaccaa 2-11-3 GAtaatattgcacCAA 217_1 5297 -16 218 agataatattgcacca 2-12-2 AGataatattgcacCA 218_1 5298 -16 219 gccaagaagataatat 2-10-4 GCcaagaagataATAT 219_1 5305 -17 220 cacagccacataaact 4-10-2 CACAgccacataaaCT 220_1 5406 -21 221 ttgtaattgtggaaac 2-12-2 TTgtaattgtggaaAC 221_1 5463 -12 222 tgacttgtaattgtgg 2-11-3 TGacttgtaattgTGG 222_1 5467 -18 223 tctaactgaaatagtc 2-12-2 TCtaactgaaatagTC 223_1 5503 -13 224 gtggttctaactgaaa 3-11-2 GTGgttctaactgaAA 224_1 5508 -16 225 caatatgggacttggt 2-12-2 CAatatgggacttgGT 225_1 5522 -18 226 atgacaatatgggact 3-11-2 ATGacaatatgggaCT 226_1 5526 -17 227 tatgacaatatgggac 4-10-2 TATGacaatatgggAC 227_1 5527 -17 228 atatgacaatatggga 4-10-2 ATATgacaatatggGA 228_1 5528 -17 229 cttcacttaataatta 2-11-3 CTtcacttaataaTTA 229_1 5552 -13 230 ctgcttcacttaataa 4-10-2 CTGCttcacttaatAA 230_1 5555 -18 231 aagactgcttcactta 2-11-3 AAgactgcttcacTTA 231_1 5559 -17 232 gaatgccctaattatg 4-10-2 GAATgccctaattaTG 232_1 5589 -19 233 tggaatgccctaatta 3-11-2 TGGaatgccctaatTA 233_1 5591 -19 234 gcaaatgccagtaggt 3-11-2 GCAaatgccagtagGT 234_1 5642 -23 235 ctaatggaaggatttg 3-11-2 CTAatggaaggattTG 235_1 5673 -15 236 aatatagaacctaatg 2-12-2 AAtatagaacctaaTG 236_1 5683 -10 237 gaaagaatagaatgtt 3-10-3 GAAagaatagaatGTT 237_1 5769 -12 238 atgggtaatagattat 3-11-2 ATGggtaatagattAT 238_1 5893 -15 239 gaaagagcacagggtg 2-12-2 GAaagagcacagggTG 239_1 6103 -18 240 ctacatagagggaatg 4-10-2 CTACatagagggaaTG 240_1 6202 -18 241 gcttcctacatagagg 2-10-4 GCttcctacataGAGG 241_1 6207 -24 242 tgcttcctacatagag 4-10-2 TGCTtcctacatagAG 242_1 6208 -22 243 tgggcttgaaatatgt 2-11-3 TGggcttgaaataTGT 243_1 6417 -19 244 cattatatttaagaac 3-11-2 CATtatatttaagaAC 244_1 6457 -11 245 tcggttatgttatcat 2-10-4 TCggttatgttaTCAT 245_1 6470 -19 246 cactttatctggtcgg 2-10-4 CActttatctggTCGG 246_1 6482 -22 247 aaattggcacagcgtt 3-10-3 AAAttggcacagcGTT 247_1 6505 -18 248 accgtgacagtaaatg 4-9-3 ACCGtgacagtaaATG 248_1 6577 -20 249 tgggaaccgtgacagta 2-13-2 TGggaaccgtgacagTA 249_1 6581 -22 250 ccacatataggtcctt 2-11-3 CCacatataggtcCTT 250_1 6597 -21 251 catattgctaccatac 2-11-3 CAtattgctaccaTAC 251_1 6617 -18 252 tcatattgctaccata 3-10-3 TCAtattgctaccATA 252_1 6618 -19 253 caattgtcatattgct 4-8-4 CAATtgtcatatTGCT 253_1 6624 -21 254 cattcaattgtcatattg 3-12-3 CATtcaattgtcataTTG 254_1 6626 -18 255 tttctactgggaatttg 4-9-4 TTTCtactgggaaTTTG 255_1 6644 -20 256 caattagtgcagccag 3-10-3 CAAttagtgcagcCAG 256_1 6672 -21 257 gaataatgttcttatcc 4-10-3 GAATaatgttcttaTCC 257_1 6704 -20 258 cacaaattgaataatgttct 4-13-3 CACAaattgaataatgtTCT 258_1 6709 -20 259 catgcacaaattgaataat 4-11-4 CATGcacaaattgaaTAAT 259_1 6714 -20 260 atcctgcaatttcacat 3-11-3 ATCctgcaatttcaCAT 260_1 6832 -22 261 ccaccatagctgatca 2-12-2 CCaccatagctgatCA 261_1 6868 -22 262 accaccatagctgatca 2-12-3 ACcaccatagctgaTCA 262_1 6868 -23 263 caccaccatagctgatc 2-13-2 CAccaccatagctgaTC 263_1 6869 -21 264 tagtcggcaccaccat 2-12-2 TAgtcggcaccaccAT 264_1 6877 -22 265 cttgtagtcggcaccac 1-14-2 CttgtagtcggcaccAC 265_1 6880 -21 266 cttgtagtcggcacca 1-13-2 CttgtagtcggcacCA 266_1 6881 -21 267 cgcttgtagtcggcac 2-12-2 CGcttgtagtcggcAC 267_1 6883 -21 268 tcaataaagatcaggc 3-11-2 TCAataaagatcagGC 268_1 6942 -17 269 tggacttacaagaatg 2-12-2 TGgacttacaagaaTG 269_1 6986 -14 270 atggacttacaagaat 3-11-2 ATGgacttacaagaAT 270_1 6987 -15 271 gctcaagaaattggat 4-10-2 GCTCaagaaattggAT 271_1 7073 -19 272 tactgtagaacatggc 4-10-2 TACTgtagaacatgGC 272_1 7133 -21 273 gcaattcatttgatct 4-9-3 GCAAttcatttgaTCT 273_1 7239 -20 274 tgaagggaggagggacac 2-14-2 TGaagggaggagggacAC 274_1 7259 -20 275 agtggtgaagggaggag 2-13-2 AGtggtgaagggaggAG 275_1 7265 -21 276 tagtggtgaagggaggag 2-14-2 TAgtggtgaagggaggAG 276_1 7265 -21 277 atagtggtgaagggaggag 1-16-2 AtagtggtgaagggaggAG 277_1 7265 -20 278 tagtggtgaagggagga 2-13-2 TAgtggtgaagggagGA 278_1 7266 -21 279 atagtggtgaagggagga 2-14-2 ATagtggtgaagggagGA 279_1 7266 -21 280 tagtggtgaagggagg 3-11-2 TAGtggtgaagggaGG 280_1 7267 -21 281 atagtggtgaagggagg 3-12-2 ATAgtggtgaagggaGG 281_1 7267 -22 282 gatagtggtgaagggagg 2-14-2 GAtagtggtgaagggaGG 282_1 7267 -21 283 atagtggtgaagggag 4-10-2 ATAGtggtgaagggAG 283_1 7268 -20 284 gatagtggtgaagggag 2-12-3 GAtagtggtgaaggGAG 284_1 7268 -21 285 gagatagtggtgaagg 2-10-4 GAgatagtggtgAAGG 285_1 7271 -20 286 catgggagatagtggt 4-10-2 CATGggagatagtgGT 286_1 7276 -22 287 acaaataatggttactct 4-10-4 ACAAataatggttaCTCT 287_1 7302 -20 288 acacacaaataatggtta 4-10-4 ACACacaaataatgGTTA 288_1 7306 -20 289 gagggacacacaaataat 3-11-4 GAGggacacacaaaTAAT 289_1 7311 -21 290 atatagagaggctcaa 4-8-4 ATATagagaggcTCAA 290_1 7390 -21 291 ttgatatagagaggct 2-10-4 TTgatatagagaGGCT 291_1 7393 -20 292 gcatttgatatagaga 4-9-3 GCATttgatatagAGA 292_1 7397 -20 293 tttgcatttgatatag 2-11-3 TTtgcatttgataTAG 293_1 7400 -15 294 ctggaagaataggttc 3-11-2 CTGgaagaataggtTC 294_1 7512 -17 295 actggaagaataggtt 4-10-2 ACTGgaagaataggTT 295_1 7513 -18 296 tactggaagaataggt 4-10-2 TACTggaagaatagGT 296_1 7514 -18 297 tggcttatcctgtact 4-10-2 TGGCttatcctgtaCT 297_1 7526 -25 298 atggcttatcctgtac 2-10-4 ATggcttatcctGTAC 298_1 7527 -22 299 tatggcttatcctgta 4-10-2 TATGgcttatcctgTA 299_1 7528 -22 300 gtatggcttatcctgt 3-10-3 GTAtggcttatccTGT 300_1 7529 -23 301 atgaatatatgcccagt 2-11-4 ATgaatatatgccCAGT 301_1 7547 -22 302 gatgaatatatgccca 2-10-4 GAtgaatatatgCCCA 302_1 7549 -22 303 caagatgaatatatgcc 3-10-4 CAAgatgaatataTGCC 303_1 7551 -21 304 gacaacatcagtataga 4-9-4 GACAacatcagtaTAGA 304_1 7572 -22 305 caagacaacatcagta 4-8-4 CAAGacaacatcAGTA 305_1 7576 -20 306 cactcctagttccttt 3-10-3 CACtcctagttccTTT 306_1 7601 -22 307 aacactcctagttcct 3-10-3 AACactcctagttCCT 307_1 7603 -22 308 taacactcctagttcc 2-11-3 TAacactcctagtTCC 308_1 7604 -20 309 ctaacactcctagttc 2-12-2 CTaacactcctagtTC 309_1 7605 -18 310 tgataacataactgtg 2-12-2 TGataacataactgTG 310_1 7637 -13 311 ctgataacataactgt 2-10-4 CTgataacataaCTGT 311_1 7638 -18 312 tttgaactcaagtgac 4-10-2 TTTGaactcaagtgAC 312_1 7654 -16 313 tcctttacttagctag 4-9-3 TCCTttacttagcTAG 313_1 7684 -23 314 gagtttggattagctg 2-11-3 GAgtttggattagCTG 314_1 7764 -20 315 tgggatatgacaggga 2-11-3 TGggatatgacagGGA 315_1 7838 -21 316 tgtgggatatgacagg 4-10-2 TGTGggatatgacaGG 316_1 7840 -22 317 atatggaagggatatc 4-10-2 ATATggaagggataTC 317_1 7875 -17 318 acaggatatggaaggg 3-10-3 ACAggatatggaaGGG 318_1 7880 -21 319 atttcaacaggatatgg 4-9-4 ATTTcaacaggatATGG 319_1 7885 -20 320 gagtaatttcaacagg 2-11-3 GAgtaatttcaacAGG 320_1 7891 -17 321 agggagtaatttcaaca 4-9-4 AGGGagtaatttcAACA 321_1 7893 -22 322 attagggagtaatttca 4-9-4 ATTAgggagtaatTTCA 322_1 7896 -21 323 cttactattagggagt 2-10-4 CTtactattaggGAGT 323_1 7903 -20 324 cagcttactattaggg 2-11-3 CAgcttactattaGGG 324_1 7906 -20 326 atttcagcttactattag 3-11-4 ATTtcagcttactaTTAG 326_1 7908 -20 325 tcagcttactattagg 3-10-3 TCAgcttactattAGG 325_1 7907 -20 327 ttcagcttactattag 2-10-4 TTcagcttactaTTAG 327_1 7908 -17 328 cagatttcagcttact 4-10-2 CAGAtttcagcttaCT 328_1 7913 -21 329 gactacaactagaggg 3-11-2 GACtacaactagagGG 329_1 7930 -19 330 agactacaactagagg 4-10-2 AGACtacaactagaGG 330_1 7931 -19 331 aagactacaactagag 2-12-2 AAgactacaactagAG 331_1 7932 -13 332 atgatttaattctagtcaaa 4-12-4 ATGAtttaattctagtCAAA 332_1 7982 -20 333 tttaattctagtcaaa 3-10-3 TTTaattctagtcAAA 333_1 7982 -12 334 gatttaattctagtca 4-8-4 GATTtaattctaGTCA 334_1 7984 -20 771 tgatttaattctagtca 3-10-4 TGAtttaattctaGTCA 771_1 7984 -20 335 atgatttaattctagtca 4-11-3 ATGAtttaattctagTCA 335_1 7984 -20 336 gatgatttaattctagtca 4-13-2 GATGatttaattctagtCA 336_1 7984 -20 337 gatttaattctagtca 2-10-4 GAtttaattctaGTCA 337_1 7984 -18 338 gatgatttaattctagtc 4-11-3 GATGatttaattctaGTC 338_1 7985 -20 339 tgatttaattctagtc 2-12-2 TGatttaattctagTC 339_1 7985 -13 340 gagatgatttaattcta 4-9-4 GAGAtgatttaatTCTA 340_1 7988 -20 341 gagatgatttaattct 3-10-3 GAGatgatttaatTCT 341_1 7989 -16 342 cagattgatggtagtt 4-10-2 CAGAttgatggtagTT 342_1 8030 -19 343 ctcagattgatggtag 2-10-4 CTcagattgatgGTAG 343_1 8032 -20 344 gttagccctcagattg 3-10-3 GTTagccctcagaTTG 344_1 8039 -23 345 tgtattgttagccctc 2-10-4 TGtattgttagcCCTC 345_1 8045 -24 346 acttgtattgttagcc 2-10-4 ACttgtattgttAGCC 346_1 8048 -22 347 agccagtatcagggac 3-11-2 AGCcagtatcagggAC 347_1 8191 -23 348 ttgacaatagtggcat 2-10-4 TTgacaatagtgGCAT 348_1 8213 -20 349 acaagtggtatcttct 3-10-3 ACAagtggtatctTCT 349_1 8228 -19 350 aatctactttacaagt 4-10-2 AATCtactttacaaGT 350_1 8238 -16 351 cacagtagatgcctgata 2-12-4 CAcagtagatgcctGATA 351_1 8351 -24 352 gaacacagtagatgcc 2-11-3 GAacacagtagatGCC 352_1 8356 -21 353 cttggaacacagtagat 4-11-2 CTTGgaacacagtagAT 353_1 8359 -20 354 atatcttggaacacag 3-10-3 ATAtcttggaacaCAG 354_1 8364 -18 355 tctttaatatcttggaac 3-11-4 TCTttaatatcttgGAAC 355_1 8368 -19 356 tgatttctttaatatcttg 2-13-4 TGatttctttaatatCTTG 356_1 8372 -19 357 tgatgatttctttaatatc 2-13-4 TGatgatttctttaaTATC 357_1 8375 -18 358 aggctaagtcatgatg 3-11-2 AGGctaagtcatgaTG 358_1 8389 -19 359 ttgatgaggctaagtc 4-10-2 TTGAtgaggctaagTC 359_1 8395 -19 360 ccaggattatactctt 3-11-2 CCAggattatactcTT 360_1 8439 -20 361 gccaggattatactct 2-10-4 GCcaggattataCTCT 361_1 8440 -23 362 ctgccaggattatact 3-11-2 CTGccaggattataCT 362_1 8442 -21 363 cagaaacttatactttatg 4-13-2 CAGAaacttatactttaTG 363_1 8473 -19 364 aagcagaaacttatact 4-9-4 AAGCagaaacttaTACT 364_1 8478 -20 365 gaagcagaaacttatact 3-11-4 GAAgcagaaacttaTACT 365_1 8478 -20 366 tggaagcagaaacttatact 3-15-2 TGGaagcagaaacttataCT 366_1 8478 -21 367 tggaagcagaaacttatac 3-13-3 TGGaagcagaaacttaTAC 367_1 8479 -20 368 aagcagaaacttatac 2-11-3 AAgcagaaacttaTAC 368_1 8479 -13 369 tggaagcagaaacttata 3-11-4 TGGaagcagaaactTATA 369_1 8480 -21 370 aagggatattatggag 4-10-2 AAGGgatattatggAG 370_1 8587 -18 371 tgccggaagatttcct 2-12-2 TGccggaagatttcCT 371_1 8641 -21 372 atggattgggagtaga 4-10-2 ATGGattgggagtaGA 372_1 8772 -21 373 agatggattgggagta 2-12-2 AGatggattgggagTA 373_1 8774 -18 374 aagatggattgggagt 3-11-2 AAGatggattgggaGT 374_1 8775 -18 375 acaagatggattggga 2-10-4 ACaagatggattGGGA 375_1 8777 -20 375 acaagatggattggga 2-12-2 ACaagatggattggGA 375_2 8777 -17 376 agaaggttcagacttt 3-9-4 AGAaggttcagaCTTT 376_1 8835 -20 377 gcagaaggttcagact 2-11-3 GCagaaggttcagACT 377_1 8837 -21 377 gcagaaggttcagact 3-11-2 GCAgaaggttcagaCT 377_2 8837 -22 378 tgcagaaggttcagac 4-10-2 TGCAgaaggttcagAC 378_1 8838 -22 379 agtgcagaaggttcag 2-11-3 AGtgcagaaggttCAG 379_1 8840 -20 379 agtgcagaaggttcag 4-10-2 AGTGcagaaggttcAG 379_2 8840 -21 380 aagtgcagaaggttca 4-10-2 AAGTgcagaaggttCA 380_1 8841 -20 381 taagtgcagaaggttc 2-10-4 TAagtgcagaagGTTC 381_1 8842 -19 382 tctaagtgcagaaggt 2-10-4 TCtaagtgcagaAGGT 382_1 8844 -21 383 ctcaggagttctacttc 3-12-2 CTCaggagttctactTC 383_1 8948 -20 384 ctcaggagttctactt 3-10-3 CTCaggagttctaCTT 384_1 8949 -21 385 atggaggtgactcaggag 1-15-2 AtggaggtgactcaggAG 385_1 8957 -20 386 atggaggtgactcagga 2-13-2 ATggaggtgactcagGA 386_1 8958 -21 387 atggaggtgactcagg 2-11-3 ATggaggtgactcAGG 387_1 8959 -21 388 tatggaggtgactcagg 2-12-3 TAtggaggtgactcAGG 388_1 8959 -21 389 atatggaggtgactcagg 2-14-2 ATatggaggtgactcaGG 389_1 8959 -21 390 tatggaggtgactcag 4-10-2 TATGgaggtgactcAG 390_1 8960 -21 391 atatggaggtgactcag 2-11-4 ATatggaggtgacTCAG 391_1 8960 -22 392 catatggaggtgactcag 2-14-2 CAtatggaggtgactcAG 392_1 8960 -20 393 atatggaggtgactca 3-10-3 ATAtggaggtgacTCA 393_1 8961 -20 394 catatggaggtgactca 2-12-3 CAtatggaggtgacTCA 394_1 8961 -21 395 catatggaggtgactc 2-10-4 CAtatggaggtgACTC 395_1 8962 -20 396 gcatatggaggtgactc 2-13-2 GCatatggaggtgacTC 396_1 8962 -21 397 tgcatatggaggtgactc 2-14-2 TGcatatggaggtgacTC 397_1 8962 -21 398 ttgcatatggaggtgactc 1-16-2 TtgcatatggaggtgacTC 398_1 8962 -20 399 tttgcatatggaggtgactc 1-17-2 TttgcatatggaggtgacTC 399_1 8962 -21 400 gcatatggaggtgact 2-12-2 GCatatggaggtgaCT 400_1 8963 -20 401 tgcatatggaggtgact 2-13-2 TGcatatggaggtgaCT 401_1 8963 -20 402 ttgcatatggaggtgact 3-13-2 TTGcatatggaggtgaCT 402_1 8963 -22 403 tttgcatatggaggtgact 1-16-2 TttgcatatggaggtgaCT 403_1 8963 -20 404 tgcatatggaggtgac 3-11-2 TGCatatggaggtgAC 404_1 8964 -20 405 ttgcatatggaggtgac 3-11-3 TTGcatatggaggtGAC 405_1 8964 -21 406 tttgcatatggaggtgac 4-12-2 TTTGcatatggaggtgAC 406_1 8964 -21 407 tttgcatatggaggtga 4-11-2 TTTGcatatggaggtGA 407_1 8965 -21 408 tttgcatatggaggtg 2-10-4 TTtgcatatggaGGTG 408_1 8966 -21 409 aagtgaagttcaacagc 2-11-4 AAgtgaagttcaaCAGC 409_1 8997 -20 410 tgggaagtgaagttca 2-10-4 TGggaagtgaagTTCA 410_1 9002 -20 411 atgggaagtgaagttc 2-11-3 ATgggaagtgaagTTC 411_1 9003 -17 412 gatgggaagtgaagtt 4-9-3 GATGggaagtgaaGTT 412_1 9004 -21 413 ctgtgatgggaagtgaa 3-11-3 CTGtgatgggaagtGAA 413_1 9007 -20 414 attgagtgaatccaaa 3-10-3 ATTgagtgaatccAAA 414_1 9119 -14 415 aattgagtgaatccaa 2-10-4 AAttgagtgaatCCAA 415_1 9120 -16 416 gataattgagtgaatcc 4-10-3 GATAattgagtgaaTCC 416_1 9122 -20 417 gtgataattgagtgaa 3-10-3 GTGataattgagtGAA 417_1 9125 -16 418 aagaaaggtgcaataa 3-10-3 AAGaaaggtgcaaTAA 418_1 9155 -14 419 caagaaaggtgcaata 2-10-4 CAagaaaggtgcAATA 419_1 9156 -15 420 acaagaaaggtgcaat 4-10-2 ACAAgaaaggtgcaAT 420_1 9157 -16 421 atttaaactcacaaac 2-12-2 ATttaaactcacaaAC 421_1 9171 -10 422 ctgttaggttcagcga 2-10-4 CTgttaggttcaGCGA 422_1 9235 -24 423 tctgaatgaacatttcg 4-9-4 TCTGaatgaacatTTCG 423_1 9260 -20 424 ctcattgaaggttctg 2-10-4 CTcattgaaggtTCTG 424_1 9281 -20 425 ctaatctcattgaagg 3-11-2 CTAatctcattgaaGG 425_1 9286 -17 426 cctaatctcattgaag 2-12-2 CCtaatctcattgaAG 426_1 9287 -16 427 actttgatctttcagc 3-10-3 ACTttgatctttcAGC 427_1 9305 -20 428 actatgcaacactttg 2-12-2 ACtatgcaacacttTG 428_1 9315 -15 429 caaatagctttatcgg 3-10-3 CAAatagctttatCGG 429_1 9335 -17 430 ccaaatagctttatcg 2-10-4 CCaaatagctttATCG 430_1 9336 -19 431 tccaaatagctttatc 4-10-2 TCCAaatagctttaTC 431_1 9337 -18 432 gatccaaatagcttta 4-10-2 GATCcaaatagcttTA 432_1 9339 -18 433 atgatccaaatagctt 2-10-4 ATgatccaaataGCTT 433_1 9341 -19 434 tatgatccaaatagct 4-10-2 TATGatccaaatagCT 434_1 9342 -18 435 taaacagggctgggaat 4-9-4 TAAAcagggctggGAAT 435_1 9408 -22 436 acttaaacagggctgg 2-10-4 ACttaaacagggCTGG 436_1 9412 -21 437 acacttaaacagggct 2-10-4 ACacttaaacagGGCT 437_1 9414 -22 438 gaacacttaaacaggg 4-8-4 GAACacttaaacAGGG 438_1 9416 -20 439 agagaacacttaaacag 4-9-4 AGAGaacacttaaACAG 439_1 9418 -20 440 ctacagagaacactta 4-8-4 CTACagagaacaCTTA 440_1 9423 -20 441 atgctacagagaacact 3-10-4 ATGctacagagaaCACT 441_1 9425 -22 442 ataaatgctacagagaaca 4-11-4 ATAAatgctacagagAACA 442_1 9427 -20 443 agataaatgctacagaga 2-12-4 AGataaatgctacaGAGA 443_1 9430 -20 444 tagagataaatgctaca 4-9-4 TAGAgataaatgcTACA 444_1 9434 -21 445 tagatagagataaatgct 4-11-3 TAGAtagagataaatGCT 445_1 9437 -20 446 caatatactagatagaga 4-10-4 CAATatactagataGAGA 446_1 9445 -21 447 tacacaatatactagatag 4-11-4 TACAcaatatactagATAG 447_1 9448 -21 448 ctacacaatatactag 3-10-3 CTAcacaatatacTAG 448_1 9452 -16 449 gctacacaatatacta 4-8-4 GCTAcacaatatACTA 449_1 9453 -21 450 atatgctacacaatatac 4-10-4 ATATgctacacaatATAC 450_1 9455 -20 451 tgatatgctacacaat 4-8-4 TGATatgctacaCAAT 451_1 9459 -20 452 atgatatgatatgctac 4-9-4 ATGAtatgatatgCTAC 452_1 9464 -21 453 gaggagagagacaataaa 4-10-4 GAGGagagagacaaTAAA 453_1 9495 -20 454 ctaggaggagagagaca 3-11-3 CTAggaggagagagACA 454_1 9500 -22 455 tattctaggaggagaga 4-10-3 TATTctaggaggagAGA 455_1 9504 -21 456 ttatattctaggaggag 4-10-3 TTATattctaggagGAG 456_1 9507 -21 457 gtttatattctaggag 3-9-4 GTTtatattctaGGAG 457_1 9510 -20 458 tggagtttatattctagg 2-12-4 TGgagtttatattcTAGG 458_1 9512 -22 459 cgtaccaccactctgc 2-11-3 CGtaccaccactcTGC 459_1 9590 -25 460 tgaggaaatcattcattc 4-10-4 TGAGgaaatcattcATTC 460_1 9641 -22 461 tttgaggaaatcattcat 4-10-4 TTTGaggaaatcatTCAT 461_1 9643 -20 462 aggctaatcctatttg 4-10-2 AGGCtaatcctattTG 462_1 9657 -22 463 tttaggctaatcctat 4-8-4 TTTAggctaatcCTAT 463_1 9660 -22 464 tgctccagtgtaccct 3-11-2 TGCtccagtgtaccCT 464_1 9755 -27 465 tagtagtactcgatag 2-10-4 TAgtagtactcgATAG 465_1 9813 -18 466 ctaattgtagtagtactc 3-12-3 CTAattgtagtagtaCTC 466_1 9818 -20 467 tgctaattgtagtagt 2-10-4 TGctaattgtagTAGT 467_1 9822 -19 468 agtgctaattgtagta 4-10-2 AGTGctaattgtagTA 468_1 9824 -19 469 gcaagtgctaattgta 4-10-2 GCAAgtgctaattgTA 469_1 9827 -20 470 gaggaaatgaactaattta 4-13-2 GAGGaaatgaactaattTA 470_1 9881 -18 471 caggaggaaatgaacta 4-11-2 CAGGaggaaatgaacTA 471_1 9886 -19 472 ccctagagtcatttcc 2-11-3 CCctagagtcattTCC 472_1 9902 -24 473 atcttacatgatgaagc 3-11-3 ATCttacatgatgaAGC 473_1 9925 -20 475 agacacactcagatttcag 2-15-2 AGacacactcagatttcAG 475_1 9967 -20 474 gacacactcagatttcag 3-13-2 GACacactcagatttcAG 474_1 9967 -20 476 aagacacactcagatttcag 3-15-2 AAGacacactcagatttcAG 476_1 9967 -21 477 agacacactcagatttca 2-13-3 AGacacactcagattTCA 477_1 9968 -20 478 aagacacactcagatttca 3-13-3 AAGacacactcagattTCA 478_1 9968 -21 479 aaagacacactcagatttca 2-14-4 AAagacacactcagatTTCA 479_1 9968 -20 480 gaaagacacactcagatttc 3-14-3 GAAagacacactcagatTTC 480_1 9969 -20 481 aagacacactcagatttc 4-11-3 AAGAcacactcagatTTC 481_1 9969 -21 482 aaagacacactcagatttc 4-11-4 AAAGacacactcagaTTTC 482_1 9969 -20 483 tgaaagacacactcagattt 4-14-2 TGAAagacacactcagatTT 483_1 9970 -20 484 tgaaagacacactcagatt 2-13-4 TGaaagacacactcaGATT 484_1 9971 -21 485 tgaaagacacactcagat 3-12-3 TGAaagacacactcaGAT 485_1 9972 -20 486 attgaaagacacactca 4-10-3 ATTGaaagacacacTCA 486_1 9975 -19 487 tcattgaaagacacact 2-11-4 TCattgaaagacaCACT 487_1 9977 -18 488 ttccatcattgaaaga 3-9-4 TTCcatcattgaAAGA 488_1 9983 -18 489 ataataccacttatcat 4-9-4 ATAAtaccacttaTCAT 489_1 10010 -20 490 ttacttaatttctttgga 2-12-4 TTacttaatttcttTGGA 490_1 10055 -20 491 ttagaactagctttatca 3-12-3 TTAgaactagctttaTCA 491_1 10101 -20 492 gaggtacaaatatagg 3-10-3 GAGgtacaaatatAGG 492_1 10171 -18 493 cttatgatacaactta 3-10-3 CTTatgatacaacTTA 493_1 10384 -15 494 tcttatgatacaactt 2-11-3 TCttatgatacaaCTT 494_1 10385 -15 495 ttcttatgatacaact 3-11-2 TTCttatgatacaaCT 495_1 10386 -15 496 cagtttcttatgatac 2-11-3 CAgtttcttatgaTAC 496_1 10390 -16 497 gcagtttcttatgata 3-11-2 GCAgtttcttatgaTA 497_1 10391 -19 498 tacaaatgtctattaggtt 4-12-3 TACAaatgtctattagGTT 498_1 10457 -21 499 tgtacaaatgtctattag 4-11-3 TGTAcaaatgtctatTAG 499_1 10460 -20 500 agcatcacaattagta 3-11-2 AGCatcacaattagTA 500_1 10535 -18 501 ctaatgatagtgaagc 3-11-2 CTAatgatagtgaaGC 501_1 10548 -17 502 agctaatgatagtgaa 3-11-2 AGCtaatgatagtgAA 502_1 10550 -16 503 atgccttgacatatta 4-10-2 ATGCcttgacatatTA 503_1 10565 -20 504 ctcaagattattgacac 4-9-4 CTCAagattattgACAC 504_1 10623 -20 505 acctcaagattattga 2-10-4 ACctcaagattaTTGA 505_2 10626 -18 505 acctcaagattattga 3-9-4 ACCtcaagattaTTGA 505_1 10626 -20 506 aacctcaagattattg 4-10-2 AACCtcaagattatTG 506_1 10627 -17 507 cacaaacctcaagattatt 4-13-2 CACAaacctcaagattaTT 507_1 10628 -20 508 gtacttaattagacct 3-9-4 GTActtaattagACCT 508_1 10667 -21 509 agtacttaattagacc 4-9-3 AGTActtaattagACC 509_1 10668 -20 510 gtatgaggtggtaaac 4-10-2 GTATgaggtggtaaAC 510_1 10688 -18 511 aggaaacagcagaagtg 2-11-4 AGgaaacagcagaAGTG 511_1 10723 -21 512 gcacaacccagaggaa 2-12-2 GCacaacccagaggAA 512_1 10735 -20 513 caagcacaacccagag 3-11-2 CAAgcacaacccagAG 513_1 10738 -20 514 ttcaagcacaacccag 3-10-3 TTCaagcacaaccCAG 514_1 10740 -21 515 aattcaagcacaaccc 2-10-4 AAttcaagcacaACCC 515_1 10742 -20 516 taataattcaagcacaacc 4-13-2 TAATaattcaagcacaaCC 516_1 10743 -20 517 actaataattcaagcac 4-9-4 ACTAataattcaaGCAC 517_1 10747 -20 518 ataatactaataattcaagc 4-12-4 ATAAtactaataattcAAGC 518_1 10749 -19 519 tagatttgtgaggtaa 2-10-4 TAgatttgtgagGTAA 519_1 11055 -18 520 agccttaattctccat 4-10-2 AGCCttaattctccAT 520_1 11091 -24 521 aatgatctagagcctta 4-9-4 AATGatctagagcCTTA 521_1 11100 -22 522 ctaatgatctagagcc 3-10-3 CTAatgatctagaGCC 522_1 11103 -22 523 actaatgatctagagc 3-9-4 ACTaatgatctaGAGC 523_1 11104 -21 524 cattaacatgttcttatt 3-11-4 CATtaacatgttctTATT 524_1 11165 -19 525 acaagtacattaacatgttc 4-12-4 ACAAgtacattaacatGTTC 525_1 11170 -22 526 ttacaagtacattaacatg 4-11-4 TTACaagtacattaaCATG 526_1 11173 -20 527 gctttattcatgtttat 4-9-4 GCTTtattcatgtTTAT 527_1 11195 -22 528 gctttattcatgttta 3-11-2 GCTttattcatgttTA 528_1 11196 -18 529 agagctttattcatgttt 3-13-2 AGAgctttattcatgtTT 529_1 11197 -20 530 ataagagctttattcatg 4-10-4 ATAAgagctttattCATG 530_1 11200 -21 531 cataagagctttattca 4-9-4 CATAagagctttaTTCA 531_1 11202 -21 532 agcataagagctttat 4-8-4 AGCAtaagagctTTAT 532_1 11205 -22 533 tagattgtttagtgca 3-10-3 TAGattgtttagtGCA 533_1 11228 -20 534 gtagattgtttagtgc 2-10-4 GTagattgtttaGTGC 534_1 11229 -21 535 gacaattctagtagatt 4-9-4 GACAattctagtaGATT 535_1 11238 -21 536 ctgacaattctagtag 3-9-4 CTGacaattctaGTAG 536_1 11241 -20 537 gctgacaattctagta 4-10-2 GCTGacaattctagTA 537_1 11242 -21 538 aggattaagatacgta 2-12-2 AGgattaagatacgTA 538_1 11262 -15 539 caggattaagatacgt 2-11-3 CAggattaagataCGT 539_1 11263 -17 540 tcaggattaagatacg 3-11-2 TCAggattaagataCG 540_1 11264 -16 541 ttcaggattaagatac 2-10-4 TTcaggattaagATAC 541_1 11265 -15 542 aggaagaaagtttgattc 4-10-4 AGGAagaaagtttgATTC 542_1 11308 -21 543 tcaaggaagaaagtttga 4-10-4 TCAAggaagaaagtTTGA 543_1 11311 -20 544 ctcaaggaagaaagtttg 4-10-4 CTCAaggaagaaagTTTG 544_1 11312 -20 545 tgctcaaggaagaaagt 3-10-4 TGCtcaaggaagaAAGT 545_1 11315 -21 546 aattatgctcaaggaaga 4-11-3 AATTatgctcaaggaAGA 546_1 11319 -20 547 taggataccacattatga 4-12-2 TAGGataccacattatGA 547_1 11389 -22 548 cataatttattccattcctc 2-15-3 CAtaatttattccattcCTC 548_1 11449 -22 549 tgcataatttattccat 4-10-3 TGCAtaatttattcCAT 549_1 11454 -22 550 actgcataatttattcc 4-10-3 ACTGcataatttatTCC 550_1 11456 -21 551 ctaaactgcataatttatt 4-11-4 CTAAactgcataattTATT 551_1 11458 -20 552 ataactaaactgcata 2-10-4 ATaactaaactgCATA 552_1 11465 -16 553 ttattaataactaaactgc 3-12-4 TTAttaataactaaaCTGC 553_1 11468 -19 554 tagtacattattaataact 4-13-2 TAGTacattattaataaCT 554_1 11475 -18 555 cataactaaggacgtt 4-10-2 CATAactaaggacgTT 555_1 11493 -17 556 tcataactaaggacgt 2-11-3 TCataactaaggaCGT 556_1 11494 -16 557 cgtcataactaaggac 4-10-2 CGTCataactaaggAC 557_1 11496 -17 558 tcgtcataactaagga 2-12-2 TCgtcataactaagGA 558_1 11497 -16 559 atcgtcataactaagg 2-10-4 ATcgtcataactAAGG 559_1 11498 -17 560 gttagtatcttacatt 2-11-3 GTtagtatcttacATT 560_1 11525 -15 561 ctctattgttagtatc 3-10-3 CTCtattgttagtATC 561_1 11532 -17 562 agtatagagttactgt 3-10-3 AGTatagagttacTGT 562_1 11567 -19 563 ttcctggtgatacttt 4-10-2 TTCCtggtgatactTT 563_1 11644 -21 564 gttcctggtgatactt 4-10-2 GTTCctggtgatacTT 564_1 11645 -21 565 tgttcctggtgatact 2-12-2 TGttcctggtgataCT 565_1 11646 -20 566 ataaacatgaatctctcc 2-12-4 ATaaacatgaatctCTCC 566_1 11801 -20 567 ctttataaacatgaatctc 3-12-4 CTTtataaacatgaaTCTC 567_1 11804 -19 568 ctgtctttataaacatg 3-10-4 CTGtctttataaaCATG 568_1 11810 -19 569 ttgttataaatctgtctt 2-12-4 TTgttataaatctgTCTT 569_1 11820 -18 570 ttaaatttattcttggata 3-12-4 TTAaatttattcttgGATA 570_1 11849 -19 571 cttaaatttattcttgga 2-12-4 CTtaaatttattctTGGA 571_1 11851 -19 572 cttcttaaatttattcttg 4-13-2 CTTCttaaatttattctTG 572_1 11853 -18 573 tatgtttctcagtaaag 4-9-4 TATGtttctcagtAAAG 573_1 11877 -19 574 gaattatctttaaacca 3-10-4 GAAttatctttaaACCA 574_1 11947 -18 575 cccttaaatttctaca 3-11-2 CCCttaaatttctaCA 575_1 11980 -20 576 acactgctcttgtacc 4-10-2 ACACtgctcttgtaCC 576_1 11995 -23 577 tgacaacactgctctt 3-10-3 TGAcaacactgctCTT 577_1 12000 -21 578 tacatttattgggctc 4-10-2 TACAtttattgggcTC 578_1 12081 -19 579 gtacatttattgggct 2-10-4 GTacatttattgGGCT 579_1 12082 -23 580 ttggtacatttattgg 3-10-3 TTGgtacatttatTGG 580_1 12085 -18 581 catgttggtacatttat 4-10-3 CATGttggtacattTAT 581_1 12088 -21 582 aatcatgttggtacat 4-10-2 AATCatgttggtacAT 582_1 12092 -16 583 aaatcatgttggtaca 2-12-2 AAatcatgttggtaCA 583_1 12093 -14 584 gacaagtttggattaa 3-11-2 GACaagtttggattAA 584_1 12132 -14 585 aatgttcagatgcctc 2-10-4 AAtgttcagatgCCTC 585_1 12197 -21 586 gcttaatgttcagatg 2-12-2 GCttaatgttcagaTG 586_1 12201 -17 587 cgtacatagcttgatg 4-10-2 CGTAcatagcttgaTG 587_1 12267 -20 588 gtgaggaattaggata 3-11-2 GTGaggaattaggaTA 588_1 12753 -17 589 gtaacaatatggtttg 3-11-2 GTAacaatatggttTG 589_1 12780 -15 590 gaaatattgtagacta 2-11-3 GAaatattgtagaCTA 590_1 13151 -14 591 ttgaaatattgtagac 3-11-2 TTGaaatattgtagAC 591_1 13153 -12 592 aagtctagtaatttgc 2-10-4 AAgtctagtaatTTGC 592_1 13217 -17 593 gctcagtagattataa 4-10-2 GCTCagtagattatAA 593_1 13259 -17 594 catacactgttgctaa 3-10-3 CATacactgttgcTAA 594_1 13296 -19 595 atggtctcaaatcatt 3-10-3 ATGgtctcaaatcATT 595_1 13314 -17 596 caatggtctcaaatca 4-10-2 CAATggtctcaaatCA 596_1 13316 -18 597 ttcctattgattgact 4-10-2 TTCCtattgattgaCT 597_1 13568 -20 598 tttctgttcacaacac 4-10-2 TTTCtgttcacaacAC 598_1 13600 -17 599 aggaacccactaatct 2-11-3 AGgaacccactaaTCT 599_1 13702 -20 600 taaatggcaggaaccc 3-11-2 TAAatggcaggaacCC 600_1 13710 -19 601 gtaaatggcaggaacc 4-10-2 GTAAatggcaggaaCC 601_1 13711 -20 602 ttgtaaatggcaggaa 2-11-3 TTgtaaatggcagGAA 602_1 13713 -16 603 ttatgagttaggcatg 2-10-4 TTatgagttaggCATG 603_1 13835 -19 604 ccaggtgaaactttaa 3-11-2 CCAggtgaaactttAA 604_1 13935 -17 605 cccttagtcagctcct 3-10-3 CCCttagtcagctCCT 605_1 13997 -30 606 acccttagtcagctcc 2-10-4 ACccttagtcagCTCC 606_1 13998 -27 607 cacccttagtcagctc 2-11-3 CAcccttagtcagCTC 607_1 13999 -24 608 tctcttactaggctcc 3-10-3 TCTcttactaggcTCC 608_1 14091 -24 609 cctatctgtcatcatg 2-11-3 CCtatctgtcatcATG 609_1 14178 -20 610 tcctatctgtcatcat 3-11-2 TCCtatctgtcatcAT 610_1 14179 -20 611 gagaagtgtgagaagc 3-11-2 GAGaagtgtgagaaGC 611_1 14808 -19 612 catccttgaagtttag 4-10-2 CATCcttgaagtttAG 612_1 14908 -19 613 taataagatggctccc 3-10-3 TAAtaagatggctCCC 613_1 15046 -21 614 caaggcataataagat 3-11-2 CAAggcataataagAT 614_1 15053 -14 615 ccaaggcataataaga 2-10-4 CCaaggcataatAAGA 615_1 15054 -18 616 tgatccaattctcacc 2-12-2 TGatccaattctcaCC 616_1 15151 -19 617 atgatccaattctcac 3-10-3 ATGatccaattctCAC 617_1 15152 -19 618 cgcttcatcttcaccc 3-11-2 CGCttcatcttcacCC 618_1 15260 -26 619 tatgacactgcatctt 2-10-4 TAtgacactgcaTCTT 619_1 15317 -19 620 gtatgacactgcatct 3-10-3 GTAtgacactgcaTCT 620_1 15318 -21 621 tgtatgacactgcatc 2-10-4 TGtatgacactgCATC 621_1 15319 -20 622 ttctcttctgtaagtc 4-10-2 TTCTcttctgtaagTC 622_1 15363 -19 623 ttctacagaggaacta 2-10-4 TTctacagaggaACTA 623_1 15467 -17 624 actacagttctacaga 3-10-3 ACTacagttctacAGA 624_1 15474 -19 625 ttcccacaggtaaatg 4-10-2 TTCCcacaggtaaaTG 625_1 15561 -21 626 attatttgaatatactcatt 4-12-4 ATTAtttgaatatactCATT 626_1 15594 -20 627 tgggaggaaattatttg 4-10-3 TGGGaggaaattatTTG 627_1 15606 -20 628 tgactcatcttaaatg 4-10-2 TGACtcatcttaaaTG 628_1 15621 -17 629 ctgactcatcttaaat 3-11-2 CTGactcatcttaaAT 629_1 15622 -16 630 tttactctgactcatc 3-10-3 TTTactctgactcATC 630_1 15628 -17 631 tattggaggaattatt 3-11-2 TATtggaggaattaTT 631_1 15642 -14 632 gtattggaggaattat 3-11-2 GTAttggaggaattAT 632_1 15643 -16 633 tggtatacttctctaagtat 2-15-3 TGgtatacttctctaagTAT 633_1 15655 -22 634 gatctcttggtatact 4-10-2 GATCtcttggtataCT 634_1 15666 -20 635 cagacaactctatacc 2-12-2 CAgacaactctataCC 635_1 15689 -18 636 aacatcagacaactcta 4-9-4 AACAtcagacaacTCTA 636_1 15693 -21 637 taacatcagacaactc 4-10-2 TAACatcagacaacTC 637_1 15695 -16 638 tttaacatcagacaactc 4-10-4 TTTAacatcagacaACTC 638_1 15695 -20 639 atttaacatcagacaa 2-12-2 ATttaacatcagacAA 639_1 15698 -11 640 cctatttaacatcagac 2-11-4 CCtatttaacatcAGAC 640_1 15700 -20 641 tccctatttaacatca 3-10-3 TCCctatttaacaTCA 641_1 15703 -21 642 tcaacgactattggaat 4-9-4 TCAAcgactattgGAAT 642_1 15737 -20 643 cttatattctggctat 4-9-3 CTTAtattctggcTAT 643_1 15850 -20 644 atccttatattctggc 4-10-2 ATCCttatattctgGC 644_1 15853 -23 645 gatccttatattctgg 2-10-4 GAtccttatattCTGG 645_1 15854 -21 646 tgatccttatattctg 3-10-3 TGAtccttatattCTG 646_1 15855 -19 647 attgaaacttgatcct 4-8-4 ATTGaaacttgaTCCT 647_1 15864 -21 648 actgtcattgaaactt 2-10-4 ACtgtcattgaaACTT 648_1 15870 -16 649 tcttactgtcattgaa 3-11-2 TCTtactgtcattgAA 649_1 15874 -16 650 aggatcttactgtcatt 2-11-4 AGgatcttactgtCATT 650_1 15877 -21 651 gcaaatcaactccatc 3-10-3 GCAaatcaactccATC 651_1 15896 -20 652 gtgcaaatcaactcca 3-10-3 GTGcaaatcaactCCA 652_1 15898 -22 653 caattatttctttgtgc 4-10-3 CAATtatttctttgTGC 653_1 15910 -21 654 tggcaacaattatttctt 3-11-4 TGGcaacaattattTCTT 654_1 15915 -21 655 gctggcaacaattatt 3-9-4 GCTggcaacaatTATT 655_1 15919 -21 656 atccatttctactgcc 4-10-2 ATCCatttctactgCC 656_1 15973 -24 657 taatatctattgatttcta 4-11-4 TAATatctattgattTCTA 657_1 15988 -20 658 tcaatagtgtagggca 2-12-2 TCaatagtgtagggCA 658_1 16093 -18 659 ttcaatagtgtagggc 3-11-2 TTCaatagtgtaggGC 659_1 16094 -19 660 aggttaattaattcaatag 4-11-4 AGGTtaattaattcaATAG 660_1 16102 -21 661 catttgtaatccctag 3-10-3 CATttgtaatcccTAG 661_2 16163 -20 661 catttgtaatccctag 3-9-4 CATttgtaatccCTAG 661_1 16163 -22 662 acatttgtaatcccta 3-10-3 ACAtttgtaatccCTA 662_1 16164 -20 663 aacatttgtaatccct 2-10-4 AAcatttgtaatCCCT 663_2 16165 -21 663 aacatttgtaatccct 3-9-4 AACatttgtaatCCCT 663_1 16165 -22 664 taaatttcaagttctg 2-11-3 TAaatttcaagttCTG 664_1 16184 -14 665 gtttaaatttcaagttct 3-11-4 GTTtaaatttcaagTTCT 665_1 16185 -19 666 ccaagtttaaatttcaag 4-10-4 CCAAgtttaaatttCAAG 666_1 16189 -21 667 acccaagtttaaatttc 4-9-4 ACCCaagtttaaaTTTC 667_1 16192 -22 668 catacagtgacccaagttt 2-14-3 CAtacagtgacccaagTTT 668_1 16199 -23 669 acatcccatacagtga 2-11-3 ACatcccatacagTGA 669_1 16208 -21 670 agcacagctctacatc 2-10-4 AGcacagctctaCATC 670_1 16219 -22 671 atatagcacagctcta 3-9-4 ATAtagcacagcTCTA 671_1 16223 -21 672 tccatatagcacagct 3-11-2 TCCatatagcacagCT 672_1 16226 -22 673 atttccatatagcaca 3-9-4 ATTtccatatagCACA 673_1 16229 -20 674 tttatttccatatagca 4-9-4 TTTAtttccatatAGCA 674_1 16231 -22 675 tttatttccatatagc 3-10-3 TTTatttccatatAGC 675_1 16232 -18 676 aaggagaggagattatg 4-9-4 AAGGagaggagatTATG 676_1 16409 -21 677 agttcttgtgttagct 3-11-2 AGTtcttgtgttagCT 677_1 16456 -21 678 gagttcttgtgttagc 2-12-2 GAgttcttgtgttaGC 678_1 16457 -20 679 attaattatccatccac 3-10-4 ATTaattatccatCCAC 679_1 16590 -21 680 atcaattaattatccatc 3-11-4 ATCaattaattatcCATC 680_1 16593 -19 681 agaatcaattaattatcc 3-12-3 AGAatcaattaattaTCC 681_1 16596 -18 682 tgagataccgtgcatg 2-12-2 TGagataccgtgcaTG 682_1 16656 -18 683 aatgagataccgtgca 2-10-4 AAtgagataccgTGCA 683_1 16658 -21 684 ctgtggttaggctaat 3-11-2 CTGtggttaggctaAT 684_1 16834 -19 685 aagagtaagggtctgtggtt 1-17-2 AagagtaagggtctgtggTT 685_1 16842 -21 686 gatgggttaagagtaa 4-9-3 GATGggttaagagTAA 686_1 16854 -19 687 agcagatgggttaaga 3-11-2 AGCagatgggttaaGA 687_1 16858 -20 688 tgtaaacatttgtagc 2-10-4 TGtaaacatttgTAGC 688_1 16886 -19 689 cctgcttataaatgta 3-11-2 CCTgcttataaatgTA 689_1 16898 -19 690 tgccctgcttataaat 4-10-2 TGCCctgcttataaAT 690_1 16901 -23 691 tcttcttagttcaata 2-12-2 TCttcttagttcaaTA 691_1 16935 -15 692 tggtttctaactacat 2-10-4 TGgtttctaactACAT 692_1 16980 -18 693 agtttggtttctaacta 2-12-3 AGtttggtttctaaCTA 693_1 16983 -19 694 gaatgaaacttgcctg 3-10-3 GAAtgaaacttgcCTG 694_1 17047 -18 695 attatccttacatgat 3-10-3 ATTatccttacatGAT 695_1 17173 -17 696 gtacccaattatcctt 2-11-3 GTacccaattatcCTT 696_1 17180 -21 697 tgtacccaattatcct 3-10-3 TGTacccaattatCCT 697_1 17181 -24 698 ttgtacccaattatcc 2-11-3 TTgtacccaattaTCC 698_1 17182 -20 699 tttgtacccaattatc 3-11-2 TTTgtacccaattaTC 699_1 17183 -17 700 agcagcaggttatatt 4-10-2 AGCAgcaggttataTT 700_1 17197 -22 701 tgggaagtggtctggg 3-10-3 TGGgaagtggtctGGG 701_1 17292 -25 702 ctggagagtgataata 3-11-2 CTGgagagtgataaTA 702_1 17322 -17 703 aatgctggattacgtc 4-10-2 AATGctggattacgTC 703_1 17354 -19 704 caatgctggattacgt 2-11-3 CAatgctggattaCGT 704_1 17355 -19 705 ttgttcagaagtatcc 2-10-4 TTgttcagaagtATCC 705_1 17625 -19 706 gatgatttgcttggag 2-10-4 GAtgatttgcttGGAG 706_1 17646 -21 707 gaaatcattcacaacc 3-10-3 GAAatcattcacaACC 707_1 17860 -17 708 ttgtaacatctactac 3-10-3 TTGtaacatctacTAC 708_1 17891 -16 709 cattaagcagcaagtt 3-11-2 CATtaagcagcaagTT 709_1 17923 -17 710 ttactagatgtgagca 3-11-2 TTActagatgtgagCA 710_1 17942 -18 711 tttactagatgtgagc 2-11-3 TTtactagatgtgAGC 711_1 17943 -18 712 gaccaagcaccttaca 3-11-2 GACcaagcaccttaCA 712_1 17971 -22 713 agaccaagcaccttac 3-10-3 AGAccaagcacctTAC 713_1 17972 -22 714 atgggttaaataaagg 2-10-4 ATgggttaaataAAGG 714_1 18052 -15 715 tcaaccagagtattaa 2-12-2 TCaaccagagtattAA 715_1 18067 -13 716 gtcaaccagagtatta 3-11-2 GTCaaccagagtatTA 716_1 18068 -18 717 attgtaaagctgatat 2-11-3 ATtgtaaagctgaTAT 717_1 18135 -14 718 cacataattgtaaagc 2-10-4 CAcataattgtaAAGC 718_1 18141 -16 719 gaggtctgctatttac 2-11-3 GAggtctgctattTAC 719_1 18274 -19 720 tgtagattcaatgcct 2-11-3 TGtagattcaatgCCT 720_1 18404 -20 721 cctcattatactatga 2-11-3 CCtcattatactaTGA 721_1 18456 -19 722 ccttatgctatgacac 2-12-2 CCttatgctatgacAC 722_1 18509 -18 723 tccttatgctatgaca 4-10-2 TCCTtatgctatgaCA 723_1 18510 -22 724 aagatgtttaagtata 3-10-3 AAGatgtttaagtATA 724_1 18598 -13 725 ctgattattaagatgt 2-10-4 CTgattattaagATGT 725_1 18607 -17 726 tggaaaggtatgaatt 2-12-2 TGgaaaggtatgaaTT 726_1 18808 -13 727 acttgaatggcttgga 2-12-2 ACttgaatggcttgGA 727_1 18880 -18 728 aacttgaatggcttgg 3-10-3 AACttgaatggctTGG 728_1 18881 -19 729 caatgtgttactattt 4-10-2 CAATgtgttactatTT 729_1 19004 -16 730 acaatgtgttactatt 3-10-3 ACAatgtgttactATT 730_1 19005 -15 731 catctgctatataaga 4-10-2 CATCtgctatataaGA 731_1 19063 -18 732 cctagagcaaatactt 4-10-2 CCTAgagcaaatacTT 732_1 19223 -20 733 cagagttaataataag 3-10-3 CAGagttaataatAAG 733_1 19327 -13 734 gttcaagcacaacgaa 4-10-2 GTTCaagcacaacgAA 734_1 19493 -18 735 agggttcaagcacaac 2-11-3 AGggttcaagcacAAC 735_1 19496 -18 736 tgttggagacactgtt 2-12-2 TGttggagacactgTT 736_1 19677 -17 737 aaggaggagttaggac 3-11-2 AAGgaggagttaggAC 737_1 19821 -18 738 ctatgccatttacgat 4-10-2 CTATgccatttacgAT 738_1 19884 -21 739 tcaaatgcagaattag 2-12-2 TCaaatgcagaattAG 739_1 19913 -12 740 agtgacaatcaaatgc 2-10-4 AGtgacaatcaaATGC 740_1 19921 -18 741 aagtgacaatcaaatg 2-11-3 AAgtgacaatcaaATG 741_1 19922 -12 742 gtgtaccaagtaacaa 3-11-2 GTGtaccaagtaacAA 742_1 19978 -16 743 tgggatgttaaactga 3-10-3 TGGgatgttaaacTGA 743_1 20037 -20 基序序列代表存在於寡核苷酸中之核鹼基之鄰接序列。 設計係指間隙聚體設計F-G-F’，其中每一數值代表連續經修飾核苷(例如2’修飾核苷)之數量(第一數值=5’側翼)，隨後係DNA核苷數量(第二數值=間隙區)，隨後係經修飾核苷(例如2’修飾核苷)之數量(第三數值=3’側翼)，視情況前接或後接有DNA及LNA之其他重複區域，該等重複區域未必係與靶核酸互補之鄰接序列之一部分。 寡核苷酸化合物代表基序序列之特定設計。大寫字母代表β-D-氧基LNA核苷，小寫字母代表DNA核苷，所有LNA C皆係5-甲基胞嘧啶，所有核苷間鏈接皆係硫代磷酸酯核苷間鏈接。 表6：靶向小鼠PD-L1轉錄物(SEQ ID NO: 4)之寡核苷酸、該等寡核苷酸之設計以及基於基序序列設計之特定寡核苷酸化合物(由CMP ID NO指示)。 SEQ ID NO 基序序列 設計 寡核苷酸化合物 CMP ID NO SEQ ID NO: 4 上之起點 dG 744 agtttacattttctgc 3-10-3 AGTttacattttcTGC 744_1 4189 -20 745 tatgtgaagaggagag 3-10-3 TATgtgaagaggaGAG 745_1 7797 -19 746 cacctttaaaacccca 3-10-3 CACctttaaaaccCCA 746_1 9221 -23 747 tcctttataatcacac 3-10-3 TCCtttataatcaCAC 747_1 10386 -19 748 acggtattttcacagg 3-10-3 ACGgtattttcacAGG 748_1 12389 -21 749 gacactacaatgagga 3-10-3 GACactacaatgaGGA 749_1 15088 -20 750 tggtttttaggactgt 3-10-3 TGGtttttaggacTGT 750_1 16410 -21 751 cgacaaattctatcct 3-10-3 CGAcaaattctatCCT 751_1 18688 -20 752 tgatatacaatgctac 3-10-3 TGAtatacaatgcTAC 752_1 18735 -16 753 tcgttgggtaaattta 3-10-3 TCGttgggtaaatTTA 753_1 19495 -17 754 tgctttataaatggtg 3-10-3 TGCtttataaatgGTG 754_1 19880 -19 基序序列代表存在於寡核苷酸中之核鹼基之鄰接序列。 設計係指間隙聚體設計F-G-F’，其中每一數值代表連續經修飾核苷(例如2’修飾核苷)之數量(第一數值=5’側翼)，隨後係DNA核苷數量(第二數值=間隙區)，隨後係經修飾核苷(例如2’修飾核苷)之數量(第三數值=3’側翼)，視情況前接或後接有DNA及LNA之其他重複區域，該等重複區域未必係與靶核酸互補之鄰接序列之一部分。 寡核苷酸化合物代表基序序列之特定設計。大寫字母代表β-D-氧基LNA核苷，小寫字母代表DNA核苷，所有LNA C皆係5-甲基胞嘧啶，所有核苷間鏈接皆係硫代磷酸酯核苷間鏈接。 表7：寡核苷酸基序序列及具有5’ ca生物可裂解連接體之反義化合物。 SEQ ID NO 基序序列 具有ca連接體之寡核苷酸化合物 CMP ID NO 755 caagtttacattttctgc co ao AGTttacattttcTGC 755_1 756 catatgtgaagaggagag co ao TATgtgaagaggaGAG 756_1 757 cacctttaaaacccca co ao CACctttaaaaccCCA 757_1 758 catcctttataatcacac co ao TCCtttataatcaCAC 758_1 759 caacggtattttcacagg co ao ACGgtattttcacAGG 759_1 760 cagacactacaatgagga co ao GACactacaatgaGGA 760_1 761 catggtttttaggactgt co ao TGGtttttaggacTGT 761_1 762 cacgacaaattctatcct co ao CGAcaaattctatCCT 762_1 763 catgatatacaatgctac co ao TGAtatacaatgcTAC 763_1 764 catcgttgggtaaattta co ao TCGttgggtaaatTTA 764_1 765 catgctttataaatggtg co ao TGCtttataaatgGTG 765_1 766 caacaaataatggttactct co ao ACAAataatggttaCTCT 766_1 767 cacagattgatggtagtt co ao CAGAttgatggtagTT 767_1 768 cacctatttaacatcagac co ao CCtatttaacatcAGAC 768_1 769 cactaattgtagtagtactc co ao CTAattgtagtagtaCTC 769_1 770 caataaacatgaatctctcc co ao ATaaacatgaatctCTCC 770_1 大寫字母代表β-D-氧基LNA核苷，小寫字母代表DNA核苷，所有LNA C皆係5-甲基胞嘧啶，下標o代表磷酸二酯核苷間鏈接且除非另外指示，否則其他核苷間鏈接係硫代磷酸酯核苷間鏈接。 表8：GalNAc偶聯之反義寡核苷酸化合物。 反義寡核苷酸偶聯物 CMP ID NO GN2-C6o co ao AGTttacattttcTGC 755_2 GN2-C6o co ao TATgtgaagaggaGAG 756_2 GN2-C6o co ao CACctttaaaaccCCA 757_2 GN2-C6o co ao TCCtttataatcaCAC 758_2 GN2-C6o co ao ACGgtattttcacAGG 759_2 GN2-C6o co ao GACactacaatgaGGA 760_2 GN2-C6o co ao TGGtttttaggacTGT 761_2 GN2-C6o co ao CGAcaaattctatCCT 762_2 GN2-C6o co ao TGAtatacaatgcTAC 763_2 GN2-C6o co ao TCGttgggtaaatTTA 764_2 GN2-C6o co ao TGCtttataaatgGTG 765_2 GN2-C6o co ao ACAAataatggttaCTCT 766_2 GN2-C6o co ao CAGAttgatggtagTT 767_2 GN2-C6o co ao CCtatttaacatcAGAC 768_2 GN2-C6o co ao CTAattgtagtagtaCTC 769_2 GN2-C6o co ao ATaaacatgaatctCTCC 770_2 GN2代表圖3中所展示之三價GalNAc簇，C6代表具有6個碳之胺基烷基，大寫字母代表β-D-氧基LNA核苷，小寫字母代表DNA核苷，所有LNA C皆係5-甲基胞嘧啶，下標o代表磷酸二酯核苷鏈接且除非另外指示，否則核苷間鏈接係硫代磷酸酯核苷間鏈接。代表一些分子之化學圖式展示於圖4至8中。 AAV/HBV小鼠模型 巴斯德 (Pasteur) 模型 ： 產生HLA-A2.1-/HLA-DR1-轉基因H-2種類I-/種類II-剔除(在本文中稱為HLA-A2/DR1)小鼠且在巴斯德研究院(Institut Pasteur)處飼餵。 該等小鼠代表用於人類免疫功能研究之並無任何小鼠MHC反應干擾之活體內實驗模型(Pajot等人，2004 Eur J Immunol. 34(11):3060-9)。 在該等研究中使用攜載可複製HBV DNA基因體之腺相關病毒(AAV)載體AAV血清型2/8。在無菌磷酸鹽緩衝鹽水(PBS)中稀釋AAV-HBV載體(GVPN批號6163)以達到5 × 1011 vg/mL之效價。在尾部靜脈中經靜脈內(i.v.)向小鼠注射100μL此經稀釋溶液(劑量/小鼠：5 × 1010 vg)。在攜帶HBV之小鼠之血液中檢測含有HBV DNA之完整病毒顆粒。在最多一年內檢測肝中之HBcAg以及血液中之HBV循環蛋白質HBeAg及HBsAg。在AAV2/8-HBV轉導小鼠中，HBsAg、HBeAg及HBV DNA在血清中持續至少一年(Dion等人，2013 J Virol 87:5554-5563)。 上海模型 ： 在此模型中，感染攜載HBV基因體(AAV/HBV)之重組腺相關病毒(AAV)之小鼠維持穩定病毒血症及抗原血症30週以上(Dan Yang等人，  2014 Cellular & Molecular Immunology 11, 71–78)。 自SLAC (Shanghai Laboratory Animal Center of Chinese Academy of Sciences)購買無特定病原體之雄性C57BL/6小鼠(4-6週齡)且飼養於個別通風籠中之動物護理設施中。遵循如由WuXi IACUC (Institutional Animal Care and Use Committee, WUXI IACUC方案編號R20131126-小鼠)所指示之動物護理及使用導則。使小鼠適應新環境3天且根據實驗設計進行分組。 在PBS中稀釋重組AAV-HBV，每一注射使用200 µL。此重組病毒攜載1.3個HBV基因體(基因型D，血清型ayw)之拷貝。 在第0天，經由尾部靜脈向所有小鼠注射200 µL AAV-HBV。在AAV注射之後第6、13及20天，下頜下對所有小鼠進行抽血(0.1 ml血液/小鼠)以用於收集血清。在注射後第22天，將患有穩定病毒血症之小鼠備用於寡核苷酸治療。寡核苷酸可未偶聯或偶聯GalNAc。DNA 疫苗 質體DNA無內毒素且由Plasmid-Factory (Germany)製得。pCMV-S2.S ayw編碼HBsAg (基因型D)之preS2及S結構域，且其表現由巨細胞病毒極早期基因啟動子控制(Michel等人，1995 Proc Natl Acad Sci U S A 92:5307-5311)。pCMV-HBc編碼攜載肝炎核心(HBc) Ag之HBV衣殼(Dion等人，2013 J Virol 87:5554-5563)。 如本文中所闡述使用DNA疫苗實施治療。在接種疫苗之前5天，向小鼠之肌肉中注射心臟毒素(CaTx, Latoxan refL81-02, 50 µl/肌肉)。CaTx使肌肉纖維去極化以誘導細胞退化，在注射後5天，出現新肌肉纖維且接受DNA疫苗以針對轉染獲得較佳效能。等量混合pCMV-S2.S ayw及pCMVCore (各1 mg/ml)且使小鼠在麻醉下(100 µL 12.5 mg/mL氯胺酮(ketamine)、1.25 mg/mL甲苯噻嗪(xylazine))藉由向經心臟毒素處理之脛骨前肌中進行雙側肌內注射來接受總共100 μg該混合物，如先前在Michel等人，1995 Proc Natl Acad Sci U S A 92:5307-5311中所闡述。抗 PD-L1 抗體 此係在Genetech處內部產生之小鼠抗小鼠PD-L1 IgG1抗體純系6E11。其係交叉阻斷阿替珠單抗(atezolizumab)且與在Roche處內部產生之阿替珠單抗具有類似活體外阻斷活性之替代抗體。藉由腹膜腔內(i.p.)注射在12.5 µg/g之劑量下來投與抗體。寡核苷酸合成 業內通常已知寡核苷酸合成。下文係可應用之方案。可藉由針對所使用裝置、載體及濃度略微改變之方法來產生本發明寡核苷酸。 在尿苷通用載體上使用亞磷醯胺方式在Oligomaker 48上以1 μmol規模來合成寡核苷酸。在合成結束時，使用氨水溶液在60℃下經5-16小時自固體載體裂解寡核苷酸。藉由反相HPLC (RP-HPLC)或藉由固相萃取純化寡核苷酸且藉由UPLC進行表徵，且藉由ESI-MS進一步證實分子質量。寡核苷酸之延長 : 藉由使用經5’-O-DMT保護之亞醯胺化物於乙腈之0.1 M溶液及於乙腈中之DCI (4,5-二氰基咪唑) (0.25 M)作為活化劑來偶合β-氰基乙基-亞磷醯胺(DNA-A(Bz)、DNA- G(ibu)、DNA- C(Bz)、DNA-T、LNA-5-甲基-C(Bz)L、NA-A(Bz)、LNA- G(dmf)或LNA-T)。對於最終循環而言，可使用具有期望修飾之亞磷醯胺，例如用於連接偶聯物基團之C6連接體或偶聯物基團本身。藉由使用氫化黃元素(0.01 M於9:1乙腈/吡啶中)來實施硫醇化以用於引入硫代磷酸酯鏈接。可使用於7:2:1 THF/吡啶/水中之0.02 M碘來引入磷酸二酯鏈接。其餘試劑係通常用於寡核苷酸合成者。 對於後固相合成偶聯而言，可將市售C6胺基連接體亞磷醯胺用於固相合成之最後循環中，且在去保護及自固體載體裂解之後，分離胺基連接之去保護寡核苷酸。經由使用標準合成方法活化官能基來引入偶聯物。 或者，可在仍位於固體載體上時藉由使用GalNAc-或GalNAc簇亞磷醯胺將偶聯物部分添加至寡核苷酸，如PCT/EP2015/073331或EP appl. NO. 15194811.4中所闡述。藉由 RP-HPLC 進行之純化 ： 藉由製備型RP-HPLC在Phenomenex Jupiter C18 10µ 150×10 mm管柱上來純化粗製化合物。使用0.1 M pH 8乙酸銨及乙腈作為緩衝劑且流速為5 mL/min。凍乾收集部分以得到通常白色固體形式之純化化合物。縮寫： DCI：           4,5-二氰基咪唑 DCM：         二氯甲烷 DMF：         二甲基甲醯胺 DMT：         4,4’-二甲氧基三苯甲基 THF：          四氫呋喃 Bz：             苯甲醯基 Ibu：            異丁醯基 RP-HPLC：   反相高效液相層析Tm 分析 將寡核苷酸及RNA靶(磷酸酯連接，PO)雙螺旋體在500 ml無RNase水中稀釋至3 mM且與500 ml 2×Tm 緩衝液(200mM NaCl、0.2mM EDTA、20mM pH 7.0磷酸鈉)混合。將溶液加熱至95℃保持3 min且然後將其在室溫下退火30 min。在配備有帕耳帖穩定程控器(Peltier temperature programmer) PTP6之λ 40 UV/VIS分光光度計上使用PE Templab軟體(Perkin Elmer)量測雙螺旋體熔融溫度(Tm )。將溫度自20℃斜升至95℃且然後斜降至25℃，且在260 nm下記錄吸收。使用熔融及退火之第一導數及局部最大值來評價雙螺旋體Tm 。組織特異性活體外連接體裂解分析 使用相關組織(例如肝或腎)及血清之均質物對具有擬測試生物可裂解連接體(例如DNA磷酸二酯連接體(PO連接體))之經FAM標記之寡核苷酸實施活體外裂解。 自適宜動物(例如小鼠、猴、豬或大鼠)收集組織及血清試樣且在均質化緩衝液(0.5% Igepal CA-630、25 mM pH 8.0 Tris、100 mM pH 8.0 NaCl (使用1 N NaOH調節)。向組織均質物及血清中摻加寡核苷酸直至濃度為200 µg/g組織。將試樣在37℃下培育24小時且然後使用苯酚-氯仿萃取試樣。在Dionex Ultimate 3000上使用Dionex DNApac p-100管柱及介於10mM - 1 M過氯酸鈉(pH 7.5)之間之梯度對溶液實施AIE HPLC分析。針對標準使用615 nm下螢光檢測器及260 nm下uv檢測器來測定經裂解寡核苷酸及未裂解寡核苷酸之含量。S1 核酸酶裂解分析 在S1核酸酶提取物或血清中對具有S1核酸酶易感連接體(例如DNA磷酸二酯連接體(PO連接體))之經FAM標記之寡核苷酸實施活體外裂解。 藉由S1核酸酶在核酸酶緩衝液(60 U pr.100 µL)中對100 µM寡核苷酸實施活體外裂解20分鐘及120分鐘。藉由將EDTA添加至緩衝液溶液中來停止酶促活性。在Dionex Ultimate 3000上使用Dionex DNApac p-100管柱及介於10mM - 1 M過氯酸鈉(pH 7.5)之間之梯度對溶液實施AIE HPLC分析。針對標準使用615 nm下螢光檢測器及260 nm下uv檢測器來測定經裂解寡核苷酸及未裂解寡核苷酸之含量。肝單核細胞之製備 如下文所闡述且根據由Tupin等人，2006 Methods Enzymol 417:185-201所闡述之方法(具有輕微修改)來製備來自AAV/HBV小鼠之肝臟細胞。在小鼠安樂死之後，經由肝門靜脈使用具有G25針之注射器向肝灌注10 ml無菌PBS。在器官發白時，在漢克氏平衡鹽溶液(Hank's Balanced Salt Solution，HBSS) (GIBCO® HBSS, 24020) + 5 %去補體胎牛血清(FCS)中收穫器官。經由100 μm細胞過濾器(BD Falcon, 352360)輕微壓製所收穫肝且將細胞懸浮於30 ml HBSS + 5 % FCS中。將細胞懸浮液在50 g下離心5 min。然後將上清液在289 g及4℃下離心10 min。在離心之後，棄除上清液且將糰粒在室溫下再懸浮於15 mL 35 %等滲Percoll溶液(稀釋至RPMI 1640 (GIBCO, 31870)中之GE Healthcare Percoll 17-0891-01號)且轉移至15 ml管中。將細胞在1360g及室溫下進一步離心25 min。藉由抽吸棄除上清液且使用HBSS + 5 % FCS將含有單核細胞之糰粒洗滌兩次。 在完整培養基(α-最小必需培養基(Gibco, 22571)，其補充有10 % FCS (Hyclone, SH30066號，批號APG21570)、100 U/mL青黴素(penicillin) + 100 μg/mL鏈黴素(streptomycin) + 0.3 mg/mL L-麩醯胺酸(Gibco, 10378)、1X非必須胺基酸(Gibco, 11140)、10 mM Hepes (Gibco, 15630)、1 mM丙酮酸鈉(Gibco, 11360)及50 μM β-巰基乙醇(LKB, 1830))中培養細胞。細胞之表面標記 將細胞接種於U形底96孔板中並使用PBS FACS (含有1 %牛血清白蛋白及0.01%疊氮化鈉之PBS)洗滌。將細胞與5 μL含有大鼠抗小鼠CD16/CD32抗體及可固定黃色存活標記物LD (Thermofisher, L34959)之PBS FACS在4℃下於暗處一起培育10 min。然後，使用25 μL含有針對NK P46 BV421 (大鼠Mab抗小鼠NK P46，Biolegend, 137612)及F4/80 (大鼠Mab抗小鼠F4/80 FITC, BD Biolegend, 123108)之單株抗體(Mab)之PBS FACS將細胞在4℃下於暗處染色20 min且亦添加兩種補充表面標記物：PD1 (大鼠Mab抗小鼠PD1 PE，BD Biosciences, 551892)及PDL1 (大鼠Mab抗小鼠PDL1 BV711，Biolegend, 124319)。細胞內細胞介素染色 (ICS) 分析 對脾細胞及肝單核細胞實施ICS分析。將細胞接種於U形96孔板中。將含有細胞之板在37℃下於僅完整培養基(作為陰性對照)中或與表9中所闡述之肽(在2 μg/ml之濃度下)過夜培育。在培育一小時之後添加2μg/mL佈雷菲德菌素A (Brefeldin A) (Sigma, B6542)。 在過夜培養之後，使用PBS FACS洗滌細胞且與5 μL含有大鼠抗小鼠CD16/CD32抗體及可固定黃色存活標記物LD (Thermofisher, L34959)之PBS FACS在4℃下於暗處一起培育10min。然後，使用25 μL含有Mab之PBS FACS將細胞在4℃下於暗處染色20 min。混合物係由針對CD3 (倉鼠Mab抗小鼠CD3-PerCP，BD Biosciences, 553067)、CD8 (大鼠Mab抗小鼠CD8-APC-H7，BD Biosciences, 560182)、CD4 (大鼠Mab抗小鼠CD4-PE-Cy7，BD Biosciences, 552775)及NK細胞 (大鼠Mab抗小鼠NK P46 BV421，Biolegend, 137612)之單株抗體構成。在洗滌數次之後固定細胞且使用Cytofix/Cytoperm在室溫下於暗處滲透20 min，使用Perm/Wash溶液(BD Biosciences, 554714)在4℃下洗滌。 在4℃下於暗處使用針對IFNγ (大鼠Mab抗小鼠IFNγ-APC，純系XMG1.2,BD Biosciences, 554413)及腫瘤壞死因子α (TNFα) (大鼠Mab抗小鼠TNFα-FITC，純系MP6-XT22；1/250 (BD Biosciences 554418)之抗體實施細胞內細胞介素染色30 min。在藉由流式細胞術使用MACSQuant分析儀進行分析之前，使用Perm/Wash洗滌細胞且再懸浮於含有1%甲醛之PBS FACS中。 選通CD3+CD8+CD4-及細胞CD3+CD8-CD4+且呈現於點狀圖上。定義兩個區域以選通用於每一細胞介素之陽性細胞。將該等閘門中所發現之事件數除以親代群體中之事件總數以得到反應性T細胞之百分比。對於每一小鼠而言，在單獨培養基中所獲得之百分比可視為背景且自使用肽刺激獲得之百分比扣除。 根據實驗背景來定義陽性臨限值，亦即在單獨培養基條件中針對每一組所獲得染色細胞之平均百分比+兩個標准偏差。僅代表至少5個事件之細胞介素之百分比可視為陽性。 表9：含於HBV核心蛋白及HBsAg之套膜蛋白結構域(S2+S)中之HLA-A2/DR1限制性表位。 蛋白質 起始 位置 終止 位置 序列 HLA 限制 參考文獻 核心 18 27 FLPSDFFPSV (SEQ ID NO: 773) A2 Bertoletti等人， Gastroenterology 1997;112:193-199 111 125 GRETVLEYLVSFGVW (SEQ ID NO: 774) DR1 Bertoletti等人， Gastroenterology 1997;112:193-199 套膜蛋白(S2+S) 114 128 TTFHQTLQDPRVRGL (SEQ ID NO: 775) DR1 Pajot等人，Microbes Infect 2006;8:2783-2790。 179 194 QAGFFLLTRILTIPQS (SEQ ID NO: 776) A2 + DR1 Pajot等人，Microbes Infect 2006;8:2783-2790。 183 191 FLLTRILTI (SEQ ID NO: 777) A2 Sette等人，J Immunol 1994;153:5586-5592。 200 214 TSLNFLGGTTVCLGQ (SEQ ID NO: 778) A2 + DR1 Pajot等人，Microbes Infect 2006;8:2783-2790。 204 212 FLGGTTVCL (SEQ ID NO: 779) A2 Rehermann等人，J Exp Med 1995;181: 1047-1058。 335 343 WLSLLVPFV (SEQ ID NO: 780) A2 Nayersina等人，J Immunol 1993;150: 4659-4671。 337 357 SLLVPFVQWFVGLSPTVWLSV (SEQ ID NO: 781) A2 + DR1 Loirat等人，J Immunol 2000;165: 4748-4755 348 357 GLSPTVWLSV (SEQ ID NO: 782) A2 Loirat等人，J Immunol 2000;165: 4748-4755 370 379 SILSPFLPLL (SEQ ID NO: 783) A2 Mizukoshi等人，J Immunol 2004;173: 5863-5871。 實例1 測試活體外效能  在人類PD-L1轉錄物中主要使用16至20具體間隙聚體來實施基因步移。在活體外實驗中於人類白血病單核球細胞系THP1及人類非何傑金氏K淋巴瘤(non-Hodgkin's K lymphoma)細胞系(KARPAS-299)中實施效能測試。細胞系 THP1及Karpas-299細胞系最初係購自European Collection of Authenticated Cell Cultures (ECACC)且如由供應商所推薦在37℃及5% CO2 下維持於加濕培育器中。寡核苷酸效能 將THP-1細胞(3.104 in RPMI-GLutamax, 10% FBS, 1% Pen-Strep (Thermo Fisher Scientific)添加至96孔圓底板中之寡核苷酸(4-5 ul)中且以100 µl/孔之最終體積培養6天。在一種單一濃度(20 µM)下及在自25 µM至0.004 µM之劑量範圍濃度(1:3稀釋於水中)來篩選寡核苷酸。根據製造商說明書使用MagNA Pure 96 Cellular RNA大體積套組在MagNA Pure 96系統(Roche Diagnostics)上來提取總mRNA。對於基因表現分析而言，使用TaqMan RNA-to-ct 1-Step套組(Thermo Fisher Scientific)在QuantStudio機器(Applied Biosystems)上利用靶向人類PDL1之預設計Taqman引子及用作內源性對照之ACTB (Thermo Fisher Scientific)來實施RT-qPCR。使用2(-Delta Delta C(T))方法計算相對PD-L1 mRNA表現程度且將抑制百分比(呈%形式)與對照試樣(未處理細胞)進行比較。 在RPMI 1640、2 mM麩醯胺酸及20% FBS (Sigma)中培養Karpas-299細胞。將細胞以10000個細胞/孔平鋪於96孔板中，培育24小時，然後添加溶於PBS中之寡核苷酸。單一劑量中之寡核苷酸之最終濃度為5 µM，最終培養體積為100 µl/孔或以於100 µL培養體積中自50 µM、15.8 µM、5.0 µM、1.58 µM、0.5 µM、0.158 µM、0.05 µM至0.0158 µM之劑量反應範圍來添加。在添加寡核苷酸化合物之後3天收穫細胞且根據製造商說明書使用PureLink Pro 96 RNA純化套組(Ambion)來提取RNA。根據製造商說明書使用M-MLT逆轉錄酶、隨機十聚體RETROscript、RNase抑制劑(Ambion)及100 mM dNTP組(Invitrogen，PCR等級)來合成cDNA。對於基因表現分析而言，使用TaqMan Fast Advanced Master Mix (2×) (Ambion)以雙螺旋體設置利用TaqMan引子分析針對PD-L1 (Applied Biosystems；Hs01125299_m1)及TBP (Applied Biosystems；4325803)來實施qPCR。相對PD-L1 mRNA表現程度以對照試樣(經PBS處理細胞)之%形式展示於表10中。 表10：抗PD-L1化合物在THP1及KARPAS-299細胞系中之活體外效能(來自n=3個實驗之平均值)。將PD-L1 mRNA含量正規化至KARPAS-299細胞中之TBP或THP1細胞中之ACTB且展示為對照% (經PBS處理之細胞)。 CMP ID NO KARPAS-299 細胞 5 µM CMP THP1 細胞 20 µM CMP 化合物(CMP) SEQ ID NO 1 上之起點 % mRNA of control sd % mRNA of control s d 5_1 50 1 32 11 TAattggctctacTGC 236 6_1 25 5 9 6 TCGCataagaatgaCT 371 7_1 29 2 15 5 TGaacacacagtcgCA 382 8_1 27 7 3 1 CTGaacacacagtCGC 383 9_1 23 4 11 3 TCTgaacacacagtCG 384 10_1 32 3 19 6 TTCtgaacacacagTC 385 11_1 57 5 39 16 ACaagtcatgttaCTA 463 12_1 75 5 37 12 ACacaagtcatgttAC 465 13_1 22 2 10 3 CTtacttagatgcTGC 495 14_1 33 4 23 11 ACttacttagatgCTG 496 15_1 33 7 21 6 GACttacttagatgCT 497 16_1 41 6 18 10 AGacttacttagaTGC 498 17_1 96 14 40 7 GCAggaagagactTAC 506 18_1 22 2 9 3 AATAaattccgttCAGG 541 19_1 34 6 21 9 GCAAataaattcCGTT 545 19_2 51 4 27 11 GCAaataaattccGTT 545 20_1 38 5 23 7 AGCAaataaattcCGT 546 21_1 73 8 56 15 CAGAgcaaataaatTCC 548 22_1 83 8 65 10 TGGAcagagcaaataAAT 551 23_1 86 6 80 8 ATGGacagagcaAATA 554 24_1 44 4 30 2 CAgaatggacagaGCA 558 25_1 63 10 40 11 TTCtcagaatggacAG 562 26_1 31 1 39 5 CTGAactttgacATAG 663 27_1 60 4 56 19 AAgacaaacccagacTGA 675 28_1 36 4 34 10 TATAagacaaacccAGAC 678 29_1 40 4 28 13 TTATaagacaaaccCAGA 679 30_1 30 2 18 6 TGTTataagacaaaCCC 682 31_1 77 3 67 10 TAGAacaatggtaCTTT 708 32_1 81 17 20 14 GTAGaacaatggtaCT 710 33_1 29 5 14 8 AGGtagaacaatgGTA 712 34_1 32 1 43 20 AAGAggtagaacaATGG 714 35_1 70 4 35 13 GCatccacagtaaaTT 749 36_1 83 2 66 21 GAaggttatttaaTTC 773 37_1 18 2 15 5 CTAAtcgaatgcaGCA 805 38_1 64 7 35 10 TACccaatctaatCGA 813 39_1 69 1 49 13 TAGttacccaatcTAA 817 40_1 49 5 26 9 CATttagttacccAAT 821 41_1 23 7 8 2 TCAtttagttaccCAA 822 42_1 24 6 12 3 TTcatttagttaCCCA 823 43_1 51 7 40 5 GAATtaatttcattTAGT 829 44_1 71 9 45 3 CAGTgaggaattaATTT 837 45_1 60 5 45 17 CCAAcagtgaggAATT 842 46_1 63 1 37 15 CCCaacagtgaggAAT 843 47_1 31 3 29 12 TAtacccaacagtgAGG 846 48_1 44 3 27 0 TTatacccaacagTGAG 847 49_1 38 3 26 6 TTTatacccaacagTGA 848 50_1 20 4 7 1 CCTttatacccaaCAG 851 51_1 22 3 6 2 TAACctttatacCCAA 854 52_1 28 1 29 16 AATaacctttataCCCA 855 53_1 80 11 48 10 GTAaataacctttaTA 859 54_1 54 4 37 14 ACTGtaaataacctTTAT 860 55_1 81 4 53 15 ATAtatatgcaatgAG 903 56_1 86 12 70 15 AGatatatatgcaaTG 905 57_1 56 8 27 7 GAGatatatatgcAAT 906 58_1 28 7 13 5 CCagagatatataTGC 909 59_1 88 13 69 23 CAATattccagagATAT 915 60_1 29 3 14 6 GCAAtattccagagATA 916 61_1 25 3 14 3 AGCaatattccagaGAT 917 62_1 29 4 17 2 CAGcaatattccAGAG 919 63_1 27 3 14 3 AATCagcaatattCCAG 921 64_1 23 6 12 6 ACAAtcagcaataTTCC 923 65_1 53 9 43 15 ACtaagtagttacactTCT 957 66_1 32 5 14 6 CTAAgtagttacactTC 958 67_1 35 4 31 6 GACtaagtagttacaCTT 959 68_1 64 10 55 14 TGActaagtagtTACA 962 69_1 62 11 57 16 CTTTgactaagtagTTA 964 70_1 42 9 59 13 CTCtttgactaagTAG 967 71_1 81 6 56 12 GCTCtttgactaagTA 968 72_1 27 3 39 9 CCttaaatactgtTGAC 1060 73_1 75 5 36 7 CTtaaatactgttgAC 1060 74_1 35 6 43 13 TCCttaaatactgTTG 1062 75_1 57 4 79 25 TCTCcttaaatactgTT 1063 76_1 53 6 28 6 TAtcatagttctCCTT 1073 77_1 26 4 9 2 AGTatcatagttcTCC 1075 78_1 74 5 39 12 GAgtatcatagttCTC 1076 79_1 49 5 35 6 AGagtatcatagTTCT 1077 79_2 74 6 36 8 AGAgtatcatagtTCT 1077 80_1 19 2 19 13 CAGagtatcatagTTC 1078 81_1 23 2 26 2 TTCAgagtatcataGT 1080 82_1 35 3 36 11 CTTcagagtatcATAG 1081 83_1 24 6 20 7 TTCTtcagagtatcaTA 1082 84_1 20 2 16 2 TTTcttcagagtaTCAT 1083 85_1 33 4 37 10 GAGAaaggctaagTTT 1099 86_1 42 2 35 18 GAcactcttgtaCATT 1213 87_1 50 4 54 8 TGagacactcttgtaCA 1215 88_1 50 8 28 8 TGagacactcttgTAC 1216 89_1 61 4 33 6 CTttattaaactCCAT 1266 90_1 71 8 43 12 ACCAaactttattaAA 1272 91_1 62 5 42 9 AAACctctactaagTG 1288 92_1 22 3 12 5 AGattaagacagtTGA 1310 93_1 46 3 ND ND AAgtaggagcaagaGGC 1475 94_1 42 4 60 24 AAAGtaggagcaagAGG 1476 95_1 86 15 46 10 GTtaagcagccaggAG 1806 96_1 66 6 82 27 AGggtaggatgggtAG 1842 97_1 83 19 62 36 AAGggtaggatgggTA 1843 98_1 60 9 69 5 CAAgggtaggatggGT 1844 98_2 76 13 34 7 CAagggtaggatggGT 1844 99_1 65 8 76 28 CCaagggtaggatgGG 1845 100_1 61 2 75 17 TCcaagggtaggatGG 1846 101_1 83 4 82 13 CTTCcaagggtaggAT 1848 102_1 45 3 52 14 ATCttccaagggtagGA 1849 103_1 29 2 17 7 AGaagtgatggctCATT 1936 104_1 26 3 22 1 AAGaagtgatggcTCAT 1937 105_1 34 6 22 2 GAAgaagtgatggcTCA 1938 106_1 41 5 21 5 ATGAaatgtaaacTGGG 1955 107_1 40 8 29 6 CAATgaaatgtaaaCTGG 1956 108_1 24 3 16 4 GCAAtgaaatgtaaACTG 1957 109_1 30 4 20 6 AGCAatgaaatgtaAACT 1958 110_1 44 4 34 14 GAGCaatgaaatgtAAAC 1959 111_1 18 1 13 3 TGaattcccatatcCGA 1992 112_1 69 8 35 8 AGaattatgaccaTAT 2010 113_1 77 7 38 10 AGGtaagaattatGACC 2014 114_1 97 10 56 13 TCAGgtaagaattaTGAC 2015 115_1 69 8 54 21 CTTCaggtaagaatTATG 2017 116_1 91 7 115 42 TCTTcaggtaagaATTA 2019 117_1 88 6 104 36 CTTCttcaggtaaGAAT 2021 118_1 85 6 118 17 TCTTcttcaggtaaGAA 2022 119_1 105 14 102 9 TCTtcttcaggtaAGA 2023 120_1 37 2 76 18 TGGtctaagagaaGAAG 2046 121_1 46 6 81 11 GTTGgtctaagagAAG 2049 122_1 74 11 64 4 AGTtggtctaagAGAA 2050 123_1 74 9 55 21 CAgttggtctaagAGAA 2050 124_1 65 9 95 21 GCAgttggtctaagagAA 2050 125_1 63 7 ND ND CAGTtggtctaagaGA 2051 126_1 65 6 ND ND GCagttggtctaagaGA 2051 127_1 67 14 104 34 GCagttggtctaaGAG 2052 128_1 22 6 10 3 CTcatatcagggCAGT 2063 129_1 50 4 46 9 CACAcatgttctttaAC 2087 130_1 22 4 12 12 TAAatacacacatgTTCT 2092 131_1 24 2 43 28 GTAAatacacacatgTTC 2093 132_1 33 3 20 12 TGTAaatacacacaTGTT 2094 133_1 73 17 57 21 GATCatgtaaatacACAC 2099 134_1 47 5 28 14 AGATcatgtaaataCACA 2100 135_1 35 6 26 11 CAAAgatcatgtaaatACAC 2101 136_1 30 2 14 3 ACAAagatcatgtaaaTACA 2102 137_1 52 6 24 18 GAATacaaagatcaTGTA 2108 138_1 33 5 20 6 AGAAtacaaagatcATGT 2109 139_1 37 1 22 15 CAGAatacaaagatCATG 2110 140_1 85 6 53 8 GCAGaatacaaagATCA 2112 141_1 79 4 40 6 AGGCagaatacaaagAT 2114 142_1 56 2 53 20 AAGGcagaatacaaAGA 2115 143_1 28 5 20 5 ATTagtgagggacGAA 2132 144_1 26 2 22 10 CAttagtgagggaCGA 2133 145_1 29 6 16 4 GAgggtgatggatTAG 2218 146_1 45 6 22 5 TTaggagtaataAAGG 2241 147_1 65 7 44 9 TTAatgaatttggtTG 2263 148_1 84 8 43 10 CTttaatgaatttgGT 2265 149_1 32 0 15 3 CATGgattacaactAA 2322 150_1 33 2 20 4 TCatggattacaaCTA 2323 151_1 29 1 11 3 GTCatggattacaaCT 2324 152_1 64 2 40 9 CAttaaatctagTCAT 2335 153_1 97 8 63 22 GACAttaaatctagTCA 2336 154_1 92 7 ND ND AGGGacattaaatcTA 2340 155_1 35 4 25 15 CAAAgcattataaCCA 2372 156_1 34 3 24 6 ACttactaggcaGAAG 2415 157_1 102 6 113 18 CAGAgttaactgtaCA 2545 158_1 102 10 103 15 CCAGagttaactgtAC 2546 159_1 88 7 95 18 GCcagagttaactgTA 2547 160_1 78 10 ND ND TGggccagagttaaCT 2550 161_1 59 5 26 5 CAgcatctatcagaCT 2576 162_1 78 8 42 10 TGAaataacatgagTCAT 2711 163_1 31 6 ND ND GTGaaataacatgAGTC 2713 164_1 18 2 11 3 TCTGtttatgtcacTG 2781 165_1 56 5 29 9 GTCTgtttatgtcaCT 2782 166_1 37 8 12 5 TGgtctgtttatGTCA 2784 167_1 39 1 19 3 TTGGtctgtttatgTC 2785 168_1 41 3 35 14 TCacccattgtttaAA 2842 169_1 18 3 14 4 TTcagcaaatatTCGT 2995 170_1 36 8 13 2 GTGtgttcagcaaATAT 2999 171_1 18 2 11 4 TCTattgttaggtATC 3053 172_1 67 4 26 12 ATtgcccatcttacTG 3118 173_1 71 2 33 9 TATtgcccatcttaCT 3119 174_1 47 4 20 5 AAatattgcccatCTT 3122 175_1 74 4 34 7 ATAaccttatcataCA 3174 176_1 98 19 44 12 TAtaaccttatcaTAC 3175 177_1 100 10 64 11 TTAtaaccttatcaTA 3176 178_1 72 38 28 5 TTTataaccttatCAT 3177 179_1 47 6 34 6 ACtgctattgctaTCT 3375 180_1 41 3 23 6 AGgactgctattgCTA 3378 181_1 32 6 27 7 GAGgactgctattgCT 3379 182_1 83 1 46 20 ACgtagaataataaCA 3561 183_1 94 4 52 9 CCaagtgatataATGG 3613 184_1 49 2 16 3 TTagcagaccaaGTGA 3621 185_1 96 3 26 5 GTttagcagaccaaGT 3623 186_1 78 3 46 10 TGacagtgattataTT 3856 187_1 88 5 45 21 TGTCcaagatattgAC 3868 188_1 46 6 23 6 GAAtatcctagatTGT 4066 189_1 79 3 45 14 CAaactgagaataTCC 4074 190_1 63 5 27 8 GCAaactgagaataTC 4075 191_1 77 9 37 11 TCCtattacaatcgTA 4214 192_1 74 10 36 9 TTCCtattacaatcGT 4215 193_1 91 8 51 28 ACtaatgggaggatTT 4256 194_1 95 14 67 24 TAgttcagagaataAG 4429 195_1 86 5 47 16 TAacatatagttcAGA 4436 196_1 87 4 81 20 ATAacatatagttcAG 4437 197_1 101 6 67 20 CAtaacatatagttCA 4438 198_1 91 6 60 13 TCataacatatagtTC 4439 199_1 61 3 31 10 TAGCtcctaacaatCA 4507 200_1 79 12 49 11 CTCCaatctttgtaTA 4602 201_1 74 2 58 13 TCTCcaatctttgtAT 4603 202_1 53 3 33 10 TCtatttcagccaaTC 4708 203_1 25 4 30 9 CGGaagtcagagtGAA 4782 204_1 32 5 21 7 TTAAgcatgaggaaTA 4798 205_1 34 10 26 11 TGAttgagcacctCTT 4831 206_1 81 12 62 12 GACtaattatttcgTT 4857 207_1 57 7 37 7 TGActaattatttCGT 4858 208_1 26 5 21 6 GTGactaattattTCG 4859 209_1 48 3 33 13 CTGCttgaaatgtgAC 4870 210_1 32 1 34 13 CCtgcttgaaatgTGA 4871 211_1 60 5 50 19 ATcctgcttgaaATGT 4873 212_1 111 8 110 26 ATTataaatctatTCT 5027 213_1 107 1 67 12 GCtaaatactttcATC 5151 214_1 26 3 19 6 CAttgtaacataCCTA 5251 215_1 33 2 20 4 GCattgtaacatacCT 5252 216_1 89 8 53 16 TAatattgcaccaaAT 5295 217_1 25 2 29 9 GAtaatattgcacCAA 5297 218_1 27 1 27 6 AGataatattgcacCA 5298 219_1 79 6 45 11 GCcaagaagataATAT 5305 220_1 159 16 68 14 CACAgccacataaaCT 5406 221_1 90 2 72 12 TTgtaattgtggaaAC 5463 222_1 10 2 11 5 TGacttgtaattgTGG 5467 223_1 82 1 67 18 TCtaactgaaatagTC 5503 224_1 30 1 32 9 GTGgttctaactgaAA 5508 225_1 53 7 53 15 CAatatgggacttgGT 5522 226_1 44 1 33 10 ATGacaatatgggaCT 5526 227_1 49 1 41 14 TATGacaatatgggAC 5527 228_1 77 1 54 15 ATATgacaatatggGA 5528 229_1 100 3 98 29 CTtcacttaataaTTA 5552 230_1 90 12 80 19 CTGCttcacttaatAA 5555 231_1 91 0 79 23 AAgactgcttcacTTA 5559 232_1 49 8 77 34 GAATgccctaattaTG 5589 233_1 17 7 88 33 TGGaatgccctaatTA 5591 234_1 40 5 35 10 GCAaatgccagtagGT 5642 235_1 81 6 72 25 CTAatggaaggattTG 5673 236_1 97 17 87 25 AAtatagaacctaaTG 5683 237_1 98 4 83 21 GAAagaatagaatGTT 5769 238_1 93 2 102 26 ATGggtaatagattAT 5893 239_1 110 24 44 14 GAaagagcacagggTG 6103 240_1 66 5 36 10 CTACatagagggaaTG 6202 241_1 70 4 34 8 GCttcctacataGAGG 6207 242_1 64 NA 33 6 TGCTtcctacatagAG 6208 243_1 30 NA 19 7 TGggcttgaaataTGT 6417 244_1 88 6 69 15 CATtatatttaagaAC 6457 245_1 8 2 5 2 TCggttatgttaTCAT 6470 246_1 18 9 12 4 CActttatctggTCGG 6482 247_1 37 2 19 5 AAAttggcacagcGTT 6505 248_1 46 12 29 8 ACCGtgacagtaaATG 6577 249_1 31 2 25 2 TGggaaccgtgacagTA 6581 250_1 17 2 23 9 CCacatataggtcCTT 6597 251_1 15 6 23 7 CAtattgctaccaTAC 6617 252_1 4 2 9 2 TCAtattgctaccATA 6618 253_1 65 12 85 14 CAATtgtcatatTGCT 6624 254_1 20 2 51 7 CATtcaattgtcataTTG 6626 255_1 48 8 91 41 TTTCtactgggaaTTTG 6644 256_1 11 5 23 8 CAAttagtgcagcCAG 6672 257_1 43 7 62 13 GAATaatgttcttaTCC 6704 258_1 28 2 36 19 CACAaattgaataatgtTCT 6709 259_1 64 4 78 22 CATGcacaaattgaaTAAT 6714 260_1 53 8 104 73 ATCctgcaatttcaCAT 6832 261_1 54 5 59 14 CCaccatagctgatCA 6868 262_1 42 8 52 22 ACcaccatagctgaTCA 6868 263_1 68 5 118 66 CAccaccatagctgaTC 6869 264_1 40 2 73 20 TAgtcggcaccaccAT 6877 265_1 64 6 72 35 CttgtagtcggcaccAC 6880 266_1 56 4 82 35 CttgtagtcggcacCA 6881 267_1 41 5 46 21 CGcttgtagtcggcAC 6883 268_1 51 4 33 14 TCAataaagatcagGC 6942 269_1 61 2 49 10 TGgacttacaagaaTG 6986 270_1 45 7 40 9 ATGgacttacaagaAT 6987 271_1 51 12 36 12 GCTCaagaaattggAT 7073 272_1 17 0 14 5 TACTgtagaacatgGC 7133 273_1 15 3 11 3 GCAAttcatttgaTCT 7239 274_1 64 11 ND ND TGaagggaggagggacAC 7259 275_1 52 6 50 28 AGtggtgaagggaggAG 7265 276_1 79 7 ND ND TAgtggtgaagggaggAG 7265 277_1 81 6 ND ND AtagtggtgaagggaggAG 7265 278_1 70 9 ND ND TAgtggtgaagggagGA 7266 279_1 84 9 ND ND ATagtggtgaagggagGA 7266 280_1 40 6 64 53 TAGtggtgaagggaGG 7267 281_1 42 10 ND ND ATAgtggtgaagggaGG 7267 282_1 63 7 ND ND GAtagtggtgaagggaGG 7267 283_1 27 7 38 11 ATAGtggtgaagggAG 7268 284_1 60 22 ND ND GAtagtggtgaaggGAG 7268 285_1 23 3 97 54 GAgatagtggtgAAGG 7271 286_1 51 6 72 19 CATGggagatagtgGT 7276 287_1 7 1 21 9 ACAAataatggttaCTCT 7302 288_1 66 8 48 20 ACACacaaataatgGTTA 7306 289_1 67 6 58 20 GAGggacacacaaaTAAT 7311 290_1 46 2 50 21 ATATagagaggcTCAA 7390 291_1 22 6 ND ND TTgatatagagaGGCT 7393 292_1 11 2 17 3 GCATttgatatagAGA 7397 293_1 70 18 44 8 TTtgcatttgataTAG 7400 294_1 30 1 30 9 CTGgaagaataggtTC 7512 295_1 53 5 42 10 ACTGgaagaataggTT 7513 296_1 56 2 41 15 TACTggaagaatagGT 7514 297_1 80 8 53 13 TGGCttatcctgtaCT 7526 298_1 73 6 52 14 ATggcttatcctGTAC 7527 299_1 75 7 89 25 TATGgcttatcctgTA 7528 300_1 52 5 50 11 GTAtggcttatccTGT 7529 301_1 27 3 31 6 ATgaatatatgccCAGT 7547 302_1 41 8 33 9 GAtgaatatatgCCCA 7549 303_1 8 2 ND ND CAAgatgaatataTGCC 7551 304_1 32 5 37 14 GACAacatcagtaTAGA 7572 305_1 28 5 30 23 CAAGacaacatcAGTA 7576 306_1 47 5 41 9 CACtcctagttccTTT 7601 307_1 39 6 33 7 AACactcctagttCCT 7603 308_1 68 3 42 14 TAacactcctagtTCC 7604 309_1 115 5 69 22 CTaacactcctagtTC 7605 310_1 97 16 57 14 TGataacataactgTG 7637 311_1 36 1 23 10 CTgataacataaCTGT 7638 312_1 38 5 24 5 TTTGaactcaagtgAC 7654 313_1 42 3 39 5 TCCTttacttagcTAG 7684 314_1 15 2 14 3 GAgtttggattagCTG 7764 315_1 49 28 ND ND TGggatatgacagGGA 7838 316_1 34 6 ND ND TGTGggatatgacaGG 7840 317_1 47 3 37 8 ATATggaagggataTC 7875 318_1 11 3 ND ND ACAggatatggaaGGG 7880 319_1 48 4 ND ND ATTTcaacaggatATGG 7885 320_1 18 2 16 4 GAgtaatttcaacAGG 7891 321_1 74 6 44 5 AGGGagtaatttcAACA 7893 322_1 38 5 56 28 ATTAgggagtaatTTCA 7896 323_1 66 9 32 11 CTtactattaggGAGT 7903 324_1 13 1 15 5 CAgcttactattaGGG 7906 325_1 26 4 20 9 TCAgcttactattAGG 7907 326_1 43 4 17 2 ATTtcagcttactaTTAG 7908 327_1 54 5 57 16 TTcagcttactaTTAG 7908 328_1 28 3 8 2 CAGAtttcagcttaCT 7913 329_1 43 4 37 16 GACtacaactagagGG 7930 330_1 45 12 36 10 AGACtacaactagaGG 7931 331_1 99 8 94 32 AAgactacaactagAG 7932 332_1 59 4 52 19 ATGAtttaattctagtCAAA 7982 333_1 100 2 84 23 TTTaattctagtcAAA 7982 771_1 91 9 60 19 GATTtaattctaGTCA 7984 334_1 74 6 50 5 TGAtttaattctaGTCA 7984 335_1 73 5 54 12 ATGAtttaattctagTCA 7984 336_1 15 1 26 3 GATGatttaattctagtCA 7984 337_1 71 22 49 16 GAtttaattctaGTCA 7984 338_1 43 5 30 11 GATGatttaattctaGTC 7985 339_1 98 5 90 27 TGatttaattctagTC 7985 340_1 87 21 86 2 GAGAtgatttaatTCTA 7988 341_1 92 5 85 27 GAGatgatttaatTCT 7989 342_1 7 1 7 1 CAGAttgatggtagTT 8030 343_1 7 2 24 11 CTcagattgatgGTAG 8032 344_1 3 1 14 9 GTTagccctcagaTTG 8039 345_1 14 5 20 7 TGtattgttagcCCTC 8045 346_1 10 2 11 5 ACttgtattgttAGCC 8048 347_1 52 4 52 17 AGCcagtatcagggAC 8191 348_1 33 3 18 8 TTgacaatagtgGCAT 8213 349_1 7 2 13 5 ACAagtggtatctTCT 8228 350_1 63 8 44 15 AATCtactttacaaGT 8238 351_1 36 2 ND ND CAcagtagatgcctGATA 8351 352_1 24 2 30 9 GAacacagtagatGCC 8356 353_1 23 4 103 14 CTTGgaacacagtagAT 8359 354_1 20 2 45 2 ATAtcttggaacaCAG 8364 355_1 25 3 24 6 TCTttaatatcttgGAAC 8368 356_1 39 2 41 10 TGatttctttaatatCTTG 8372 357_1 54 5 88 43 TGatgatttctttaaTATC 8375 358_1 31 4 45 27 AGGctaagtcatgaTG 8389 359_1 18 3 43 20 TTGAtgaggctaagTC 8395 360_1 6 2 11 2 CCAggattatactcTT 8439 361_1 43 5 40 14 GCcaggattataCTCT 8440 362_1 56 8 73 13 CTGccaggattataCT 8442 363_1 23 1 33 7 CAGAaacttatactttaTG 8473 364_1 49 8 45 14 AAGCagaaacttaTACT 8478 365_1 39 6 37 4 GAAgcagaaacttaTACT 8478 366_1 26 4 45 13 TGGaagcagaaacttataCT 8478 367_1 21 4 44 5 TGGaagcagaaacttaTAC 8479 368_1 97 4 70 22 AAgcagaaacttaTAC 8479 369_1 34 3 32 11 TGGaagcagaaactTATA 8480 370_1 71 7 46 19 AAGGgatattatggAG 8587 371_1 51 9 79 38 TGccggaagatttcCT 8641 372_1 45 6 52 25 ATGGattgggagtaGA 8772 373_1 27 7 30 8 AGatggattgggagTA 8774 374_1 13 3 28 6 AAGatggattgggaGT 8775 375_1 42 10 44 11 ACaagatggattGGGA 8777 375_2 41 3 45 14 ACaagatggattggGA 8777 376_1 83 9 88 32 AGAaggttcagaCTTT 8835 377_1 40 5 33 3 GCAgaaggttcagaCT 8837 377_2 28 5 20 4 GCagaaggttcagACT 8837 378_1 70 2 43 8 TGCAgaaggttcagAC 8838 379_1 23 3 55 17 AGtgcagaaggttCAG 8840 379_2 51 6 41 8 AGTGcagaaggttcAG 8840 380_1 34 6 35 7 AAGTgcagaaggttCA 8841 381_1 44 11 24 6 TAagtgcagaagGTTC 8842 382_1 37 5 45 9 TCtaagtgcagaAGGT 8844 383_1 75 5 147 26 CTCaggagttctactTC 8948 384_1 90 10 141 55 CTCaggagttctaCTT 8949 385_1 73 8 234 116 AtggaggtgactcaggAG 8957 386_1 33 4 42 7 ATggaggtgactcagGA 8958 387_1 24 3 29 14 ATggaggtgactcAGG 8959 388_1 37 2 65 15 TAtggaggtgactcAGG 8959 389_1 50 10 81 19 ATatggaggtgactcaGG 8959 390_1 42 5 61 10 TATGgaggtgactcAG 8960 391_1 36 2 76 50 ATatggaggtgacTCAG 8960 392_1 52 6 64 6 CAtatggaggtgactcAG 8960 393_1 63 5 57 6 ATAtggaggtgacTCA 8961 394_1 53 7 64 12 CAtatggaggtgacTCA 8961 395_1 51 5 56 24 CAtatggaggtgACTC 8962 396_1 23 3 41 34 GCatatggaggtgacTC 8962 397_1 34 3 54 10 TGcatatggaggtgacTC 8962 398_1 54 5 71 24 TtgcatatggaggtgacTC 8962 399_1 61 11 59 13 TttgcatatggaggtgacTC 8962 400_1 25 2 30 6 GCatatggaggtgaCT 8963 401_1 34 4 25 9 TGcatatggaggtgaCT 8963 402_1 25 4 31 20 TTGcatatggaggtgaCT 8963 403_1 51 6 37 11 TttgcatatggaggtgaCT 8963 404_1 26 1 33 5 TGCatatggaggtgAC 8964 405_1 25 2 69 19 TTGcatatggaggtGAC 8964 406_1 26 4 24 4 TTTGcatatggaggtgAC 8964 407_1 19 3 20 7 TTTGcatatggaggtGA 8965 408_1 16 5 46 16 TTtgcatatggaGGTG 8966 409_1 9 2 9 6 AAgtgaagttcaaCAGC 8997 410_1 26 8 109 52 TGggaagtgaagTTCA 9002 411_1 31 5 24 5 ATgggaagtgaagTTC 9003 412_1 49 9 19 10 GATGggaagtgaaGTT 9004 413_1 28 10 17 9 CTGtgatgggaagtGAA 9007 414_1 54 4 34 8 ATTgagtgaatccAAA 9119 415_1 11 1 14 2 AAttgagtgaatCCAA 9120 416_1 58 6 14 2 GATAattgagtgaaTCC 9122 417_1 5 1 16 3 GTGataattgagtGAA 9125 418_1 73 5 61 14 AAGaaaggtgcaaTAA 9155 419_1 86 6 64 13 CAagaaaggtgcAATA 9156 420_1 75 19 64 14 ACAAgaaaggtgcaAT 9157 421_1 75 8 50 13 ATttaaactcacaaAC 9171 422_1 21 8 23 6 CTgttaggttcaGCGA 9235 423_1 54 10 30 5 TCTGaatgaacatTTCG 9260 424_1 11 4 15 5 CTcattgaaggtTCTG 9281 425_1 87 3 52 8 CTAatctcattgaaGG 9286 426_1 95 1 85 13 CCtaatctcattgaAG 9287 427_1 31 7 22 7 ACTttgatctttcAGC 9305 428_1 64 7 49 16 ACtatgcaacacttTG 9315 429_1 18 6 21 3 CAAatagctttatCGG 9335 430_1 19 6 17 4 CCaaatagctttATCG 9336 431_1 35 4 27 8 TCCAaatagctttaTC 9337 432_1 75 8 43 7 GATCcaaatagcttTA 9339 433_1 67 11 32 8 ATgatccaaataGCTT 9341 434_1 53 5 43 6 TATGatccaaatagCT 9342 435_1 97 9 66 29 TAAAcagggctggGAAT 9408 436_1 58 12 44 17 ACttaaacagggCTGG 9412 437_1 58 10 30 12 ACacttaaacagGGCT 9414 438_1 87 38 41 3 GAACacttaaacAGGG 9416 439_1 70 4 59 33 AGAGaacacttaaACAG 9418 440_1 83 17 28 9 CTACagagaacaCTTA 9423 441_1 49 12 27 4 ATGctacagagaaCACT 9425 442_1 53 10 24 13 ATAAatgctacagagAACA 9427 443_1 23 6 20 10 AGataaatgctacaGAGA 9430 444_1 48 6 27 7 TAGAgataaatgcTACA 9434 445_1 51 3 32 8 TAGAtagagataaatGCT 9437 446_1 38 5 ND ND CAATatactagataGAGA 9445 447_1 52 3 31 1 TACAcaatatactagATAG 9448 448_1 65 6 48 11 CTAcacaatatacTAG 9452 449_1 67 9 29 2 GCTAcacaatatACTA 9453 450_1 103 17 65 15 ATATgctacacaatATAC 9455 451_1 71 13 129 22 TGATatgctacaCAAT 9459 452_1 19 4 9 1 ATGAtatgatatgCTAC 9464 453_1 75 10 45 21 GAGGagagagacaaTAAA 9495 454_1 68 6 43 10 CTAggaggagagagACA 9500 455_1 72 7 79 25 TATTctaggaggagAGA 9504 456_1 31 3 29 9 TTATattctaggagGAG 9507 457_1 38 5 62 17 GTTtatattctaGGAG 9510 458_1 15 6 15 8 TGgagtttatattcTAGG 9512 459_1 34 3 21 3 CGtaccaccactcTGC 9590 460_1 41 5 55 22 TGAGgaaatcattcATTC 9641 461_1 81 8 47 22 TTTGaggaaatcatTCAT 9643 462_1 76 8 39 5 AGGCtaatcctattTG 9657 463_1 93 12 216 12 TTTAggctaatcCTAT 9660 464_1 15 6 30 9 TGCtccagtgtaccCT 9755 465_1 27 3 25 6 TAgtagtactcgATAG 9813 466_1 9 2 7 3 CTAattgtagtagtaCTC 9818 467_1 52 3 32 6 TGctaattgtagTAGT 9822 468_1 68 11 36 16 AGTGctaattgtagTA 9824 469_1 35 6 32 3 GCAAgtgctaattgTA 9827 470_1 91 9 ND ND GAGGaaatgaactaattTA 9881 471_1 92 5 ND ND CAGGaggaaatgaacTA 9886 472_1 67 5 42 6 CCctagagtcattTCC 9902 473_1 35 5 20 8 ATCttacatgatgaAGC 9925 474_1 13 1 20 5 GACacactcagatttcAG 9967 475_1 24 4 20 2 AGacacactcagatttcAG 9967 476_1 25 4 24 7 AAGacacactcagatttcAG 9967 477_1 26 6 19 4 AGacacactcagattTCA 9968 478_1 28 4 32 13 AAGacacactcagattTCA 9968 479_1 31 8 37 6 AAagacacactcagatTTCA 9968 480_1 63 7 51 26 GAAagacacactcagatTTC 9969 481_1 37 10 ND ND AAGAcacactcagatTTC 9969 482_1 41 4 ND ND AAAGacacactcagaTTTC 9969 483_1 19 5 48 14 TGAAagacacactcagatTT 9970 484_1 60 8 68 10 TGaaagacacactcaGATT 9971 485_1 42 8 63 22 TGAaagacacactcaGAT 9972 486_1 48 9 41 20 ATTGaaagacacacTCA 9975 487_1 27 6 27 12 TCattgaaagacaCACT 9977 488_1 88 13 121 33 TTCcatcattgaAAGA 9983 489_1 80 12 ND ND ATAAtaccacttaTCAT 10010 490_1 13 4 27 15 TTacttaatttcttTGGA 10055 491_1 32 5 60 24 TTAgaactagctttaTCA 10101 492_1 58 10 55 17 GAGgtacaaatatAGG 10171 493_1 4 1 12 3 CTTatgatacaacTTA 10384 494_1 37 6 35 5 TCttatgatacaaCTT 10385 495_1 30 0 27 6 TTCttatgatacaaCT 10386 496_1 27 8 18 3 CAgtttcttatgaTAC 10390 497_1 25 10 25 6 GCAgtttcttatgaTA 10391 498_1 77 6 72 29 TACAaatgtctattagGTT 10457 499_1 66 5 69 17 TGTAcaaatgtctatTAG 10460 500_1 27 10 20 4 AGCatcacaattagTA 10535 501_1 31 10 25 5 CTAatgatagtgaaGC 10548 502_1 21 7 30 8 AGCtaatgatagtgAA 10550 503_1 35 5 39 8 ATGCcttgacatatTA 10565 504_1 64 11 79 26 CTCAagattattgACAC 10623 505_2 25 4 83 32 ACctcaagattaTTGA 10626 505_1 94 7 22 6 ACCtcaagattaTTGA 10626 506_1 31 6 34 10 AACCtcaagattatTG 10627 507_1 55 6 62 17 CACAaacctcaagattaTT 10628 508_1 66 12 40 4 GTActtaattagACCT 10667 509_1 78 5 80 10 AGTActtaattagACC 10668 510_1 36 5 42 15 GTATgaggtggtaaAC 10688 511_1 40 4 48 22 AGgaaacagcagaAGTG 10723 512_1 27 7 13 6 GCacaacccagaggAA 10735 513_1 54 5 ND ND CAAgcacaacccagAG 10738 514_1 35 7 ND ND TTCaagcacaaccCAG 10740 515_1 49 6 52 15 AAttcaagcacaACCC 10742 516_1 72 4 106 49 TAATaattcaagcacaaCC 10743 517_1 43 4 57 21 ACTAataattcaaGCAC 10747 518_1 37 3 60 12 ATAAtactaataattcAAGC 10749 519_1 9 3 6 1 TAgatttgtgagGTAA 11055 520_1 59 10 31 5 AGCCttaattctccAT 11091 521_1 41 4 34 9 AATGatctagagcCTTA 11100 522_1 34 6 34 7 CTAatgatctagaGCC 11103 523_1 52 6 52 17 ACTaatgatctaGAGC 11104 524_1 60 4 54 10 CATtaacatgttctTATT 11165 525_1 57 4 55 8 ACAAgtacattaacatGTTC 11170 526_1 53 6 44 5 TTACaagtacattaaCATG 11173 527_1 54 11 49 17 GCTTtattcatgtTTAT 11195 528_1 34 7 17 5 GCTttattcatgttTA 11196 529_1 11 2 21 4 AGAgctttattcatgtTT 11197 530_1 22 4 33 7 ATAAgagctttattCATG 11200 531_1 30 5 32 15 CATAagagctttaTTCA 11202 532_1 77 8 24 4 AGCAtaagagctTTAT 11205 533_1 8 3 15 6 TAGattgtttagtGCA 11228 534_1 4 2 10 2 GTagattgtttaGTGC 11229 535_1 41 6 33 11 GACAattctagtaGATT 11238 536_1 50 1 37 7 CTGacaattctaGTAG 11241 537_1 49 7 36 6 GCTGacaattctagTA 11242 538_1 59 2 42 11 AGgattaagatacgTA 11262 539_1 28 11 28 4 CAggattaagataCGT 11263 540_1 96 5 20 6 TCAggattaagataCG 11264 541_1 70 11 59 11 TTcaggattaagATAC 11265 542_1 53 5 28 4 AGGAagaaagtttgATTC 11308 543_1 92 13 59 12 TCAAggaagaaagtTTGA 11311 544_1 44 3 67 7 CTCAaggaagaaagTTTG 11312 545_1 43 4 32 4 TGCtcaaggaagaAAGT 11315 546_1 41 7 44 20 AATTatgctcaaggaAGA 11319 547_1 11 4 26 8 TAGGataccacattatGA 11389 548_1 25 4 26 12 CAtaatttattccattcCTC 11449 549_1 64 6 ND ND TGCAtaatttattcCAT 11454 550_1 48 17 49 7 ACTGcataatttatTCC 11456 551_1 91 10 92 15 CTAAactgcataattTATT 11458 552_1 85 8 38 9 ATaactaaactgCATA 11465 553_1 86 4 ND ND TTAttaataactaaaCTGC 11468 554_1 91 13 92 21 TAGTacattattaataaCT 11475 555_1 50 4 37 7 CATAactaaggacgTT 11493 556_1 41 5 30 7 TCataactaaggaCGT 11494 557_1 80 7 55 13 CGTCataactaaggAC 11496 558_1 86 3 59 11 TCgtcataactaagGA 11497 559_1 51 9 33 12 ATcgtcataactAAGG 11498 560_1 91 6 65 26 GTtagtatcttacATT 11525 561_1 30 3 41 8 CTCtattgttagtATC 11532 562_1 59 8 18 6 AGTatagagttacTGT 11567 563_1 65 11 41 11 TTCCtggtgatactTT 11644 564_1 57 13 45 13 GTTCctggtgatacTT 11645 565_1 57 15 30 7 TGttcctggtgataCT 11646 566_1 17 4 35 4 ATaaacatgaatctCTCC 11801 567_1 16 3 30 4 CTTtataaacatgaaTCTC 11804 568_1 60 5 45 11 CTGtctttataaaCATG 11810 569_1 20 2 19 5 TTgttataaatctgTCTT 11820 570_1 68 9 44 4 TTAaatttattcttgGATA 11849 571_1 76 8 48 12 CTtaaatttattctTGGA 11851 572_1 62 5 66 5 CTTCttaaatttattctTG 11853 573_1 28 4 44 10 TATGtttctcagtAAAG 11877 574_1 29 6 36 11 GAAttatctttaaACCA 11947 575_1 74 6 34 7 CCCttaaatttctaCA 11980 576_1 37 8 30 9 ACACtgctcttgtaCC 11995 577_1 45 14 27 6 TGAcaacactgctCTT 12000 578_1 2 1 12 5 TACAtttattgggcTC 12081 579_1 65 14 39 9 GTacatttattgGGCT 12082 580_1 34 4 53 12 TTGgtacatttatTGG 12085 581_1 41 7 35 6 CATGttggtacattTAT 12088 582_1 11 4 12 5 AATCatgttggtacAT 12092 583_1 96 16 48 9 AAatcatgttggtaCA 12093 584_1 71 15 42 13 GACaagtttggattAA 12132 585_1 46 34 39 6 AAtgttcagatgCCTC 12197 586_1 37 26 28 12 GCttaatgttcagaTG 12201 587_1 75 8 43 12 CGTAcatagcttgaTG 12267 588_1 41 10 28 5 GTGaggaattaggaTA 12753 589_1 41 5 27 9 GTAacaatatggttTG 12780 590_1 67 10 37 7 GAaatattgtagaCTA 13151 591_1 97 10 80 12 TTGaaatattgtagAC 13153 592_1 64 10 47 9 AAgtctagtaatTTGC 13217 593_1 84 7 60 9 GCTCagtagattatAA 13259 594_1 42 8 32 9 CATacactgttgcTAA 13296 595_1 101 6 79 17 ATGgtctcaaatcATT 13314 596_1 53 14 46 7 CAATggtctcaaatCA 13316 597_1 47 6 36 6 TTCCtattgattgaCT 13568 598_1 97 12 41 6 TTTCtgttcacaacAC 13600 599_1 85 1 49 11 AGgaacccactaaTCT 13702 600_1 56 3 34 7 TAAatggcaggaacCC 13710 601_1 15 4 24 8 GTAAatggcaggaaCC 13711 602_1 40 6 26 8 TTgtaaatggcagGAA 13713 603_1 59 12 26 6 TTatgagttaggCATG 13835 604_1 62 2 42 10 CCAggtgaaactttAA 13935 605_1 77 9 55 18 CCCttagtcagctCCT 13997 606_1 82 13 42 11 ACccttagtcagCTCC 13998 607_1 74 1 39 10 CAcccttagtcagCTC 13999 608_1 76 9 30 8 TCTcttactaggcTCC 14091 609_1 82 5 50 13 CCtatctgtcatcATG 14178 610_1 82 1 48 12 TCCtatctgtcatcAT 14179 611_1 41 6 50 13 GAGaagtgtgagaaGC 14808 612_1 70 5 84 19 CATCcttgaagtttAG 14908 613_1 64 14 61 16 TAAtaagatggctCCC 15046 614_1 85 2 51 14 CAAggcataataagAT 15053 615_1 47 1 35 10 CCaaggcataatAAGA 15054 616_1 74 8 53 11 TGatccaattctcaCC 15151 617_1 63 4 41 11 ATGatccaattctCAC 15152 618_1 46 7 42 9 CGCttcatcttcacCC 15260 619_1 104 4 15 4 TAtgacactgcaTCTT 15317 620_1 8 3 8 5 GTAtgacactgcaTCT 15318 621_1 21 3 27 10 TGtatgacactgCATC 15319 622_1 37 7 38 11 TTCTcttctgtaagTC 15363 623_1 49 7 36 11 TTctacagaggaACTA 15467 624_1 47 1 32 10 ACTacagttctacAGA 15474 625_1 78 8 69 6 TTCCcacaggtaaaTG 15561 626_1 70 7 ND ND ATTAtttgaatatactCATT 15594 627_1 73 7 49 25 TGGGaggaaattatTTG 15606 628_1 80 5 64 11 TGACtcatcttaaaTG 15621 629_1 71 6 66 19 CTGactcatcttaaAT 15622 630_1 31 6 41 6 TTTactctgactcATC 15628 631_1 88 2 68 18 TATtggaggaattaTT 15642 632_1 53 2 27 6 GTAttggaggaattAT 15643 633_1 23 3 39 7 TGgtatacttctctaagTAT 15655 634_1 42 9 33 3 GATCtcttggtataCT 15666 635_1 38 1 30 16 CAgacaactctataCC 15689 636_1 10 2 19 3 AACAtcagacaacTCTA 15693 637_1 13 1 11 3 TAACatcagacaacTC 15695 638_1 14 2 27 2 TTTAacatcagacaACTC 15695 639_1 101 14 81 16 ATttaacatcagacAA 15698 640_1 14 1 17 1 CCtatttaacatcAGAC 15700 641_1 65 2 ND ND TCCctatttaacaTCA 15703 642_1 41 6 42 12 TCAAcgactattgGAAT 15737 643_1 37 2 29 5 CTTAtattctggcTAT 15850 644_1 31 7 35 4 ATCCttatattctgGC 15853 645_1 13 3 8 1 GAtccttatattCTGG 15854 646_1 25 5 20 4 TGAtccttatattCTG 15855 647_1 33 6 54 10 ATTGaaacttgaTCCT 15864 648_1 43 3 27 6 ACtgtcattgaaACTT 15870 649_1 54 7 32 12 TCTtactgtcattgAA 15874 650_1 12 1 25 2 AGgatcttactgtCATT 15877 651_1 13 4 11 3 GCAaatcaactccATC 15896 652_1 10 5 16 3 GTGcaaatcaactCCA 15898 653_1 7 0 36 18 CAATtatttctttgTGC 15910 654_1 21 3 31 7 TGGcaacaattattTCTT 15915 655_1 75 9 73 24 GCTggcaacaatTATT 15919 656_1 21 6 39 6 ATCCatttctactgCC 15973 657_1 25 3 38 8 TAATatctattgattTCTA 15988 658_1 14 2 11 5 TCaatagtgtagggCA 16093 659_1 11 4 10 3 TTCaatagtgtaggGC 16094 660_1 18 1 32 12 AGGTtaattaattcaATAG 16102 661_1 33 7 25 10 CATttgtaatccCTAG 16163 661_2 64 14 31 8 CATttgtaatcccTAG 16163 662_1 48 6 34 6 ACAtttgtaatccCTA 16164 663_2 29 6 23 5 AAcatttgtaatCCCT 16165 663_1 30 6 18 6 AACatttgtaatCCCT 16165 664_1 49 1 26 6 TAaatttcaagttCTG 16184 665_1 17 3 30 10 GTTtaaatttcaagTTCT 16185 666_1 22 7 40 9 CCAAgtttaaatttCAAG 16189 667_1 89 11 ND ND ACCCaagtttaaaTTTC 16192 668_1 60 16 87 8 CAtacagtgacccaagTTT 16199 669_1 65 9 50 12 ACatcccatacagTGA 16208 670_1 83 8 103 4 AGcacagctctaCATC 16219 671_1 80 9 150 36 ATAtagcacagcTCTA 16223 672_1 57 14 ND ND TCCatatagcacagCT 16226 673_1 53 10 106 8 ATTtccatatagCACA 16229 674_1 78 3 96 14 TTTAtttccatatAGCA 16231 675_1 77 9 31 7 TTTatttccatatAGC 16232 676_1 32 6 ND ND AAGGagaggagatTATG 16409 677_1 32 5 24 6 AGTtcttgtgttagCT 16456 678_1 19 4 17 4 GAgttcttgtgttaGC 16457 679_1 14 3 25 3 ATTaattatccatCCAC 16590 680_1 11 2 20 6 ATCaattaattatcCATC 16593 681_1 31 5 40 11 AGAatcaattaattaTCC 16596 682_1 8 3 30 10 TGagataccgtgcaTG 16656 683_1 11 3 ND ND AAtgagataccgTGCA 16658 684_1 15 3 33 10 CTGtggttaggctaAT 16834 685_1 45 7 38 7 AagagtaagggtctgtggTT 16842 686_1 24 5 ND ND GATGggttaagagTAA 16854 687_1 11 2 ND ND AGCagatgggttaaGA 16858 688_1 ND ND 51 7 TGtaaacatttgTAGC 16886 689_1 83 1 54 11 CCTgcttataaatgTA 16898 690_1 103 4 73 14 TGCCctgcttataaAT 16901 691_1 104 2 64 22 TCttcttagttcaaTA 16935 692_1 ND ND 60 9 TGgtttctaactACAT 16980 693_1 ND ND 94 22 AGtttggtttctaaCTA 16983 694_1 8 2 17 5 GAAtgaaacttgcCTG 17047 695_1 98 6 51 9 ATTatccttacatGAT 17173 696_1 48 4 18 4 GTacccaattatcCTT 17180 697_1 94 2 48 9 TGTacccaattatCCT 17181 698_1 31 5 42 13 TTgtacccaattaTCC 17182 699_1 41 4 39 6 TTTgtacccaattaTC 17183 700_1 63 0 28 12 AGCAgcaggttataTT 17197 701_1 99 6 43 12 TGGgaagtggtctGGG 17292 702_1 103 2 28 5 CTGgagagtgataaTA 17322 703_1 52 6 27 9 AATGctggattacgTC 17354 704_1 67 3 37 7 CAatgctggattaCGT 17355 705_1 36 10 80 12 TTgttcagaagtATCC 17625 706_1 19 9 47 9 GAtgatttgcttGGAG 17646 707_1 44 NA 60 9 GAAatcattcacaACC 17860 708_1 46 9 32 9 TTGtaacatctacTAC 17891 709_1 56 0 79 17 CATtaagcagcaagTT 17923 710_1 30 9 46 7 TTActagatgtgagCA 17942 711_1 29 4 36 6 TTtactagatgtgAGC 17943 712_1 41 13 41 6 GACcaagcaccttaCA 17971 713_1 36 19 49 11 AGAccaagcacctTAC 17972 714_1 30 6 34 7 ATgggttaaataAAGG 18052 715_1 70 2 24 8 TCaaccagagtattAA 18067 716_1 11 4 26 8 GTCaaccagagtatTA 18068 717_1 126 56 26 6 ATtgtaaagctgaTAT 18135 718_1 73 1 42 10 CAcataattgtaAAGC 18141 719_1 23 9 55 18 GAggtctgctattTAC 18274 720_1 50 1 42 11 TGtagattcaatgCCT 18404 721_1 79 3 39 10 CCtcattatactaTGA 18456 722_1 27 6 30 8 CCttatgctatgacAC 18509 723_1 26 7 50 13 TCCTtatgctatgaCA 18510 724_1 59 1 48 12 AAGatgtttaagtATA 18598 725_1 54 2 50 13 CTgattattaagATGT 18607 726_1 92 10 84 19 TGgaaaggtatgaaTT 18808 727_1 24 8 61 16 ACttgaatggcttgGA 18880 728_1 8 4 51 14 AACttgaatggctTGG 18881 729_1 35 4 35 10 CAATgtgttactatTT 19004 730_1 36 9 53 11 ACAatgtgttactATT 19005 731_1 70 2 41 11 CATCtgctatataaGA 19063 732_1 38 NA 42 9 CCTAgagcaaatacTT 19223 733_1 102 15 15 4 CAGagttaataatAAG 19327 734_1 37 10 8 5 GTTCaagcacaacgAA 19493 735_1 13 1 38 11 AGggttcaagcacAAC 19496 736_1 49 NA 36 11 TGttggagacactgTT 19677 737_1 48 NA 32 10 AAGgaggagttaggAC 19821 738_1 36 NA 64 11 CTATgccatttacgAT 19884 739_1 105 19 66 19 TCaaatgcagaattAG 19913 740_1 44 NA 41 6 AGtgacaatcaaATGC 19921 741_1 107 NA 68 18 AAgtgacaatcaaATG 19922 742_1 102 4 27 6 GTGtaccaagtaacAA 19978 743_1 110 10 30 16 TGGgatgttaaacTGA 20037 實例2-以劑量反應曲線測試活體外效能  在實例1中所闡述之活體外效能分析中於KARPAS-299細胞中使用於PBS中之半對數連續稀釋液(50 µM、15.8µM、5.0µM、1.58µM、0.5µM、0.158µM、0.05µM至0.0158µM寡核苷酸)來測試來自表10之所選寡核苷酸。評價寡核苷酸之IC 50及最大抑制(殘餘PD-L1表現%)。 在GraphPad Prism6中實施EC50計算。IC50及最大PD-L1敲低量展示於表11中(以經對照(PBS)處理之細胞之%形式)。 表11：KARPAS-299細胞系中之最大抑制(呈鹽水之%形式)及EC50。 CMP ID NO 最大抑制 ( 殘餘PD-L1 表現% ； 經鹽水處理結果之%) EC50 (µM) 化合物 CMP SEQ ID NO: 1 上之起點 Avg SD Avg SD 6_1 11 3.3 0.69 0.11 TCGCataagaatgaCT 371 8_1 29 1.7 0.06 0.01 CTGaacacacagtCGC 383 9_1 19 1.7 0.23 0.02 TCTgaacacacagtCG 384 13_1 14 4.7 0.45 0.12 CTtacttagatgcTGC 495 41_1 10 1.8 0.19 0.02 TCAtttagttaccCAA 822 42_1 17 1.3 0.19 0.02 TTcatttagttaCCCA 823 58_1 23 1.5 0.17 0.01 CCagagatatataTGC 909 77_1 24 2.4 0.16 0.02 AGTatcatagttcTCC 1075 92_1 12 2.4 0.25 0.03 AGattaagacagtTGA 1310 111_1 3 2.0 0.27 0.03 TGaattcccatatcCGA 1992 128_1 11 1.8 0.25 0.03 CTcatatcagggCAGT 2063 151_1 16 2.7 0.28 0.05 GTCatggattacaaCT 2324 164_1 19 1.6 0.15 0.01 TCTGtttatgtcacTG 2781 166_1 36 1.7 0.11 0.02 TGgtctgtttatGTCA 2784 169_1 10 1.6 0.22 0.02 TTcagcaaatatTCGT 2995 171_1 12 2.0 0.21 0.02 TCTattgttaggtATC 3053 222_1 1 2.0 0.21 0.02 TGacttgtaattgTGG 5467 233_1 1 4.3 0.89 0.17 TGGaatgccctaatTA 5591 245_1 4 2.0 0.17 0.02 TCggttatgttaTCAT 6470 246_1 7 2.1 0.25 0.03 CActttatctggTCGG 6482 250_1 0 2.5 0.23 0.03 CCacatataggtcCTT 6597 251_1 0 2.8 0.75 0.10 CAtattgctaccaTAC 6617 252_1 3 2.2 0.19 0.02 TCAtattgctaccATA 6618 256_1 5 2.2 0.32 0.03 CAAttagtgcagcCAG 6672 272_1 1 3.2 0.69 0.10 TACTgtagaacatgGC 7133 273_1 3 2.8 0.28 0.04 GCAAttcatttgaTCT 7239 287_1 1 1.4 0.13 0.01 ACAAataatggttaCTCT 7302 292_1 2 2.1 0.21 0.02 GCATttgatatagAGA 7397 303_1 0 1.2 0.21 0.01 CAAgatgaatataTGCC 7551 314_1 3 2.1 0.39 0.04 GAgtttggattagCTG 7764 318_1 3 1.4 0.14 0.01 ACAggatatggaaGGG 7880 320_1 2 2.4 0.22 0.03 GAgtaatttcaacAGG 7891 324_1 0 2.4 0.44 0.05 CAgcttactattaGGG 7906 336_1 0 2.5 0.21 0.03 GATGatttaattctagtCA 7984 342_1 1 2.2 0.12 0.01 CAGAttgatggtagTT 8030 343_1 4 1.8 0.11 0.01 CTcagattgatgGTAG 8032 344_1 0 0.9 0.12 0.01 GTTagccctcagaTTG 8039 345_1 0 2.3 0.36 0.04 TGtattgttagcCCTC 8045 346_1 1 2.1 0.22 0.02 ACttgtattgttAGCC 8048 349_1 4 2.9 0.21 0.03 ACAagtggtatctTCT 8228 359_1 6 2.9 0.39 0.05 TTGAtgaggctaagTC 8395 360_1 0 1.7 0.18 0.02 CCAggattatactcTT 8439 374_1 5 1.7 0.33 0.03 AAGatggattgggaGT 8775 408_1 3 1.8 0.21 0.02 TTtgcatatggaGGTG 8966 409_1 0 1.8 0.21 0.02 AAgtgaagttcaaCAGC 8997 415_1 0 1.4 0.23 0.02 AAttgagtgaatCCAA 9120 417_1 7 0.9 0.15 0.01 GTGataattgagtGAA 9125 424_1 6 3.2 0.19 0.03 CTcattgaaggtTCTG 9281 429_1 5 2.5 0.48 0.05 CAAatagctttatCGG 9335 430_1 1 2.7 0.68 0.09 CCaaatagctttATCG 9336 458_1 0 4.1 0.35 0.07 TGgagtttatattcTAGG 9512 464_1 0 4.1 0.56 0.10 TGCtccagtgtaccCT 9755 466_1 1 2.1 0.21 0.02 CTAattgtagtagtaCTC 9818 474_1 0 2.4 0.27 0.03 GACacactcagatttcAG 9967 490_1 0 1.9 0.29 0.03 TTacttaatttcttTGGA 10055 493_1 3 1.8 0.20 0.02 CTTatgatacaacTTA 10384 512_1 0 3.3 0.63 0.10 GCacaacccagaggAA 10735 519_1 5 1.5 0.15 0.01 TAgatttgtgagGTAA 11055 529_1 0 2.7 0.24 0.03 AGAgctttattcatgtTT 11197 533_1 6 1.5 0.14 0.01 TAGattgtttagtGCA 11228 534_1 5 0.9 0.06 0.00 GTagattgtttaGTGC 11229 547_1 1 1.6 0.26 0.02 TAGGataccacattatGA 11389 566_1 0 3.0 0.40 0.06 ATaaacatgaatctCTCC 11801 567_1 2 2.5 0.34 0.04 CTTtataaacatgaaTCTC 11804 578_1 2 1.3 0.09 0.01 TACAtttattgggcTC 12081 582_1 1 1.6 0.20 0.02 AATCatgttggtacAT 12092 601_1 1 2.1 0.47 0.05 GTAAatggcaggaaCC 13711 619_1 4 3.4 0.44 0.08 TAtgacactgcaTCTT 15317 620_1 1 1.2 0.12 0.01 GTAtgacactgcaTCT 15318 636_1 0 1.3 0.19 0.01 AACAtcagacaacTCTA 15693 638_1 0 2.2 0.36 0.04 TAACatcagacaacTC 15695 637_1 0 2.1 0.21 0.02 TTTAacatcagacaACTC 15695 640_1 2 3.3 0.42 0.06 CCtatttaacatcAGAC 15700 645_1 1 2.9 0.34 0.04 GAtccttatattCTGG 15854 650_1 0 2.4 0.24 0.03 AGgatcttactgtCATT 15877 651_1 4 3.4 0.33 0.05 GCAaatcaactccATC 15896 652_1 0 1.3 0.16 0.01 GTGcaaatcaactCCA 15898 653_1 4 2.0 0.09 0.01 CAATtatttctttgTGC 15910 658_1 3 1.6 0.32 0.02 TCaatagtgtagggCA 16093 659_1 5 1.4 0.20 0.01 TTCaatagtgtaggGC 16094 660_1 4 2.1 0.22 0.02 AGGTtaattaattcaATAG 16102 665_1 3 1.8 0.18 0.02 GTTtaaatttcaagTTCT 16185 678_1 3 2.1 0.43 0.04 GAgttcttgtgttaGC 16457 679_1 0 3.5 0.31 0.05 ATTaattatccatCCAC 16590 680_1 4 1.6 0.12 0.01 ATCaattaattatcCATC 16593 682_1 3 2.4 0.27 0.03 TGagataccgtgcaTG 16656 683_1 0 3.2 0.16 0.03 AAtgagataccgTGCA 16658 684_1 2 2.3 0.25 0.03 CTGtggttaggctaAT 16834 687_1 5 1.3 0.13 0.01 AGCagatgggttaaGA 16858 694_1 0 1.7 0.16 0.02 GAAtgaaacttgcCTG 17047 706_1 15 3.6 0.27 0.06 GAtgatttgcttGGAG 17646 716_1 10 2.1 0.15 0.02 GTCaaccagagtatTA 18068 728_1 5 1.2 0.09 0.01 AACttgaatggctTGG 18881 733_1 0 12.7 8.01 3.62 CAGagttaataatAAG 19327 734_1 0 14.6 3.49 2.39 GTTCaagcacaacgAA 19493 735_1 0 2.5 0.30 0.04 AGggttcaagcacAAC 19496 在實例1中所闡述之活體外效能分析中在THP-1中使用1:3連續水溶液(25 µM至0.004 µM)來測試來自表6之所選寡核苷酸。評價寡核苷酸之IC 50及最大抑制(殘餘PD-L1表現百分比)。 在GraphPad Prism6中實施EC50計算。IC50及最大PD-L1敲低量展示於表12中(以經對照(PBS)處理之細胞之%形式)。 表12：THP1細胞系中之最大抑制(呈鹽水之%形式)及EC50。 CMP ID NO 最大抑制 ( 殘餘PD-L1 表現% ； 鹽水之%) EC50 (µM) 化合物 CMP SEQ ID NO: 1 上之起點 Avg SD Avg SD 6_1 12 11.5 0.73 0.38 TCGCataagaatgaCT 371 8_1 6 5.6 0.11 0.04 CTGaacacacagtCGC 383 9_1 1 14.3 0.36 0.27 TCTgaacacacagtCG 384 13_1 2 12.4 0.49 0.31 CTtacttagatgcTGC 495 41_1 14 14.6 0.38 0.27 TCAtttagttaccCAA 822 42_1 21 10.4 0.22 0.10 TTcatttagttaCCCA 823 58_1 6 19.8 0.97 0.81 CCagagatatataTGC 909 77_1 5 4.8 0.14 0.04 AGTatcatagttcTCC 1075 92_1 0 12.9 0.57 0.39 AGattaagacagtTGA 1310 128_1 15 10.1 0.23 0.13 CTcatatcagggCAGT 2063 151_1 9 14.4 0.18 0.15 GTCatggattacaaCT 2324 164_1 16 22.0 0.57 0.60 TCTGtttatgtcacTG 2781 166_1 13 11.9 0.17 0.11 TGgtctgtttatGTCA 2784 169_1 0 9.3 0.22 0.11 TTcagcaaatatTCGT 2995 171_1 11 12.9 0.28 0.20 TCTattgttaggtATC 3053 222_1 16 19.7 0.68 0.64 TGacttgtaattgTGG 5467 245_1 14 6.1 0.26 0.08 TCggttatgttaTCAT 6470 246_1 28 7.3 0.10 0.20 CActttatctggTCGG 6482 252_1 19 8.0 0.29 0.12 TCAtattgctaccATA 6618 272_1 3 9.7 0.25 0.14 TACTgtagaacatgGC 7133 314_1 13 9.6 0.31 0.15 GAgtttggattagCTG 7764 344_1 11 8.0 0.14 0.06 GTTagccctcagaTTG 8039 349_1 12 12.5 0.18 0.14 ACAagtggtatctTCT 8228 415_1 11 9.6 0.26 0.12 AAttgagtgaatCCAA 9120 493_1 15 16.5 0.48 0.34 CTTatgatacaacTTA 10384 512_1 43 14.1 0.31 0.68 GCacaacccagaggAA 10735 519_1 9 12.2 0.45 0.26 TAgatttgtgagGTAA 11055 533_1 11 13.6 0.29 0.21 TAGattgtttagtGCA 11228 534_1 9 6.5 0.09 0.03 GTagattgtttaGTGC 11229 582_1 0 12.3 0.33 0.23 AATCatgttggtacAT 12092 619_1 8 10.4 0.32 0.18 TAtgacactgcaTCTT 15317 620_1 12 24.6 1.10 1.08 GTAtgacactgcaTCT 15318 638_1 2 5.4 0.00 0.00 TAACatcagacaacTC 15695 645_1 20 29.6 1.10 1.50 GAtccttatattCTGG 15854 651_1 0 11.2 0.14 0.09 GCAaatcaactccATC 15896 658_1 11 13.8 0.48 0.32 TCaatagtgtagggCA 16093 659_1 0 8.2 0.11 0.06 TTCaatagtgtaggGC 16094 733_1 0 69.6 11.03 26.95 CAGagttaataatAAG 19327 734_1 36 16.8 2.84 2.12 GTTCaagcacaacgAA 19493 表7及8中之結果亦展示於圖2中(與其靶向SEQ ID NO: 1之PD-L1 mRNA前體之位置相關)。 自此圖可看到，幾乎所有化合物皆具有低於1 µM之EC50值及低於25%之對照細胞(經鹽水處理)中之PD-L1表現程度之靶敲低。 實例3 -聚(I:C)誘導之小鼠中使用裸及GalNAc偶聯之PD-L1反義寡核苷酸之活體外功效及效能及活體內PD-L1降低  在活體外實驗之劑量-反應研究中於MCP-11細胞中使用表6中之寡核苷酸來實施效能及功效測試。在聚(I:C)誘導之C57BL/6J雌性小鼠中於活體內測試相同寡核苷酸以及GalNAc偶聯形式(表8 CMP ID NO 755_2 - 765_2)降低PD-L1 mRNA及蛋白質表現之能力。活體外分析 將懸浮於DMEM (Sigma目錄編號D0819，補充有10%馬血清、2 mM L-麩醯胺酸、0.025 mg/ml慶大黴素(gentamicin)及1 mM丙酮酸鈉)中之MCP-11細胞(最初購自ATCC)以8000細胞/孔之密度添加至96孔圓底板中之寡核苷酸(10µl)中且以200 µl/孔之最終體積在37℃及5% CO2 下於加濕培育器中培養3天。以劑量範圍濃度(50 µM、15.8 µM、5.0 µM、1.58 µM、0.5 µM、0.158 µM、0.05 µM及0.0158 µM)篩選寡核苷酸。 根據製造商說明書使用PureLink Pro 96 RNA純化套組(Ambion)來提取總RNA。根據製造商說明書使用M-MLT逆轉錄酶、隨機十聚體RETROscript、RNase抑制劑(Ambion)及100 mM dNTP組(Invitrogen，PCR等級)來合成cDNA。對於基因表現分析，使用TaqMan Fast Advanced Master Mix (2×) (Ambion)以雙螺旋體設置利用TaqMan引子分析針對PD-L1 (Thermo Fisher Scientific；FAM-MGB Mm00452054-m1)及Gusb (Thermo Fisher Scientific; VIC-MGB-PL  Mm01197698-m1)來實施qPCR。相對PD-L1 mRNA表現程度以殘餘PD-L1表現%於PBS對照試樣(經PBS處理細胞)之%示於表9中。在GraphPad Prism6中實施EC50計算。EC50及最大PD-L1敲低量以對照(PBS)細胞之%示於表13中。 活體內分析 向C57BL/6J雌性小鼠(20-23 g；5隻小鼠/組)經皮下注射5 mg/kg靶向小鼠PD-L1之未偶聯寡核苷酸或2.8 mg/kg靶向小鼠PD-L1之GalNAc偶聯之寡核苷酸。3天後，小鼠經靜脈內注射10 mg/kg聚(I:C) (LWM, Invivogen)。在聚(I:C)注射之後5 h將小鼠處死且將肝試樣置於RNAlater (Thermo Fisher Scientific)中用於RNA提取或冷凍於乾冰上用於蛋白質提取。 根據製造商說明書使用PureLink Pro 96 RNA純化套組(Ambion)自均質化肝試樣來提取總RNA。根據製造商說明書使用M-MLT逆轉錄酶、隨機十聚體RETROscript、RNase抑制劑(Ambion)及100 mM dNTP組(Invitrogen，PCR等級)來合成cDNA。對於基因表現分析，使用TaqMan® Fast Advanced Master Mix TaqMan Fast Advanced Master Mix (2×) (Ambion)以雙螺旋體設置利用TaqMan引子分析針對PD-L1 mRNA (Thermo Fisher Scientific；FAM-MGB Mm00452054-m1)及TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1)來實施qPCR。相對PD-L1 mRNA表現程度示於表13中(以使用鹽水及聚(I:C)注射之小鼠之對照試樣之%)。 藉由在2 ml/100 mg組織之T-PER®組織蛋白提取試劑(Thermo Fisher Scientific) (與1× Halt蛋白酶抑制劑混合劑混合，無EDTA (Thermo Fisher Scientific))中均質化肝試樣來製備肝均質物。根據製造商說明書使用Coomassie Plus (Bradford)分析試劑(Thermo Scientific)來量測肝均質物中之蛋白質濃度。在4-12% Bis-Tris Plus聚丙烯醯胺凝膠(Thermo Fisher Scientific)上於1×MOPS運行緩衝液中分離肝均質物(40 µg蛋白質)且根據製造商說明書使用iBLOT乾燥印漬系統(Thermo Fisher Scientific)轉移至硝基纖維素膜中。在64 kDa帶處將每一印漬水平切割成兩個部分。在含有5%脫脂奶粉及0.05% Tween20之TBS中阻斷後，將膜與兔單株抗紐蛋白(Abcam目錄編號ab129002，以1:10000稀釋於含有5%脫脂奶粉及0.05% Tween20之TBS中，上膜)或山羊多株抗mPD-L1 (R&D Systems目錄編號AF1019，以1:1000稀釋，下膜)一起在4℃下於培育過夜。在含有0.05% Tween20之TBS中洗滌膜，且在室溫下暴露與HRP偶聯之豬抗兔IgG (DAKO，以1:3000稀釋於含有5%脫脂奶粉及0.05% Tween20之TBS中，上膜)或HRP偶聯之兔抗山羊IgG (DAKO，以1:2000稀釋) 1 h。在洗滌膜後，使用ECL select (Amersham GE Healthcare)檢測反應性。對於使用寡核苷酸治療之每一組小鼠而言，藉由與注射有鹽水及聚(I:C) (對照)之小鼠之PD-L1/紐蛋白帶強度進行比較來評估與紐蛋白帶相關之PD-L1帶的強度。結果展示於表13中，且使用裸寡核苷酸及偶聯寡核苷酸之對之西方印漬展示於圖9 A-E中。 表13：靶向小鼠PD-L1之寡核苷酸之活體外及活體內效能 CMP ID NO 化合物 CMP 最大抑制(PBS%) EC50 (µM) PD-L1 mRNA ( 對照 %) PD-L1 蛋白 ( 相對於對照 ) 744_1 AGTttacattttcTGC 9.1 0.56 86 ++ 746_1 CACctttaaaaccCCA 5.0 0.46 181 nd 747_1 TCCtttataatcaCAC 4.4 0.52 104 ++ 748_1 ACGgtattttcacAGG 1.8 0.26 102 +++ 749_1 GACactacaatgaGGA 7.6 1.21 104 nd 750_1 TGGtttttaggacTGT 12.4 0.74 84 nd 751_1 CGAcaaattctatCCT 9.9 0.69 112 nd 752_1 TGAtatacaatgcTAC 10.5 1.11 142 +++ 753_1 TCGttgggtaaatTTA 5.7 0.53 116 +++ 754_1 TGCtttataaatgGTG 5.2 0.35 98 nd 755_2 5'-GN2-C6-caAGTttacattttcTGC nd nd 58 + 757_2 5'-GN2-C6-caCACctttaaaaccCCA nd nd 62 nd 758_2 5'-GN2-C6-caTCCtttataatcaCAC nd nd 53 + 759_2 5'-GN2-C6-caACGgtattttcacAGG nd nd 66 + 760_2 5'-GN2-C6-caGACactacaatgaGGA nd nd 101 nd 761_2 5'-GN2-C6-caTGGtttttaggacTGT nd nd 99 nd 762_2 5'-GN2-C6-caCGAcaaattctatCCT nd nd 84 nd 763_2 5'-GN2-C6-caTGAtatacaatgcTAC nd nd 93 +++ 764_2 5'-GN2-C6-caTCGttgggtaaatTTA nd nd 53 + 765_2 5'-GN2-C6-caTGCtttataaatgGTG nd nd 106 nd +++：類似於對照之PD-L1/紐蛋白帶強度；++：弱於對照之PD-L1/紐蛋白帶強度；+：遠弱於對照之PD-L1/紐蛋白帶強度；nd=未測定。 自表13中之數據可看到，寡核苷酸之GalNAc偶聯明顯改良活體內PD-L1降低。mRNA之降低通常與PD-L1蛋白之降低相關。除CMP ID NO: 754_1外，在寡核苷酸偶聯至GalNAc後，低活體外EC50值通常反映良好活體內PD-L1 mRNA降低。 實例4 -來自聚(I:C)誘導之小鼠中之經分選肝細胞及非實質細胞中之活體內PK/PD  在自聚(I:C)誘導之小鼠分離之肝細胞及非實質細胞中探究裸寡核苷酸及GalNAc偶聯之寡核苷酸之分佈以及PD-L1 mRNA降低。 向C57BL/6J雌性小鼠(n=3隻/組)經皮下注射靶向小鼠PD-L1 mRNA之5 mg/kg未偶聯寡核苷酸(748_1)或7 mg/kg GalNAc偶聯之寡核苷酸(759_2)。2天後，向小鼠經腹膜腔內注射15 mg/kg聚(I:C) (LWM, Invivogen)。在聚(I:C)注射之後將小鼠麻醉18-20 h且使用含有15 mM Hepes及0.38 mM EGTA之漢克氏平衡鹽溶液經由腔靜脈以7 ml/min之流速灌注肝5 min，隨後使用膠原酶溶液(含有0.17 mg/ml膠原酶類型2 (Worthington 4176)、0.03% BSA、3.2 mM CaCl2 及1.6 g/l NaHCO3 之漢克氏平衡鹽溶液)灌注12 min。在灌注後，取出肝且打開肝囊，經由70 µm細胞過濾器使用威廉E培養基(William E medium)過濾肝懸浮液且取出細胞懸浮液之等分試樣(=混合肝臟細胞)以用於隨後分析。將其餘細胞懸浮液在50×g下離心3 min。收集上清液以用於非實質細胞之隨後純化。將糰粒再懸浮於25 ml威廉E培養基(Sigma目錄編號W1878，補充有1× Pen/Strep、2 mM L-麩醯胺酸及10% FBS (ATCC 30-2030號))中，與含有90% percoll之25 ml威廉E培養基混合且藉由在50×g下離心10 min來沈澱肝細胞。在威廉E培養基中洗滌2次後，將沈澱之肝細胞再懸浮於威廉E培養基中。將含有非實質細胞之上清液在500×g下離心7 min且將細胞再懸浮於4 ml RPMI培養基中並經由兩層percoll (25%及50% percoll)在1800×g下離心30 min。在收集兩個percoll層之間之非實質細胞後，洗滌細胞且再懸浮於RPMI培養基中。 根據製造商說明書使用PureLink Pro 96 RNA純化套組(Ambion)自純化肝細胞、非實質細胞及總肝懸浮液(未分級分離之肝臟細胞)來提取總RNA。根據製造商說明書使用M-MLT逆轉錄酶、隨機十聚體RETROscript、RNase抑制劑(Ambion)及100 mM dNTP組(Invitrogen，PCR等級)來合成cDNA。對於基因表現分析而言，使用TaqMan Fast Advanced Master Mix (2×) (Ambion)以雙螺旋體設置利用TaqMan引子分析針對PD-L1 (Thermo Fisher Scientific；FAM-MGB Mm00452054-m1)及TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1)。相對PD-L1 mRNA表現程度展示於表10中(以來自使用鹽水及聚(I:C)注射之小鼠之對照試樣之%形式)。 使用ELISA採用利用序列5´-TACCGT-s-Bio-3'之生物素化捕獲探針及利用序列5´- DIG-C12-S1-CCTGTG - 3´之地高辛偶聯檢測探針來實施寡核苷酸含量分析。該等探針僅由具有磷酸二酯主鏈之LNA組成。將肝試樣(大約50 mg)在含有一種5mm不銹鋼珠粒之2 mL埃彭道夫管(Eppendorf tube)中於1.4 mL MagNa純裂解緩衝液(Roche目錄編號03604721001)中均質化。將試樣在Retsch MM400均質器(Merck Eurolab)上均質化直至獲得均勻裂解物為止。將試樣在室溫下培育30 min。藉由將未偶聯反義寡核苷酸化合物(CMP ID NO 748_1)以界定濃度摻加至未處理肝試樣中且將其處理為試樣來生成標準。選擇摻加濃度以匹配預期試樣寡核苷酸含量(在約10倍內)。 將均質化試樣在5 × SSCT緩衝液(750 mM NaCl及75 mM檸檬酸鈉，含有0.05 % (v/v) Tween-20，pH 7.0)中稀釋最少10倍且使用捕獲-檢測溶液(於5×SSCT緩衝液中之35 nM捕獲探針及35 nM檢測探針)製備經6次2倍稀釋之一系列稀釋液並在室溫下培育30 min。將試樣轉移至96孔鏈黴抗生物素蛋白(Streptavidin)塗覆板(Nunc目錄編號436014)中且使每孔含有100 µL。將板在室溫及輕微攪動下培育1小時。使用2 × SSCT緩衝液洗滌三次且向每一孔中添加以1:4000稀釋於PBST (磷酸鹽緩衝鹽水，含有0.05 % (v/v) Tween-20，pH 7.2，新製)中之100 μL抗DIG-AP Fab片段(Roche Applied Science，目錄編號11 093 274 910)，並在室溫及輕微攪動下培育1小時。使用2 × SSCT緩衝液洗滌三次且添加100 μL鹼性磷酸酶(AP)受質溶液(藍磷基質(Blue Phos Substrate)，KPL產品代碼50-88-00，新製)。在培育30分鐘之後於輕微攪動下以分光光度方式在615 nm下量測色彩強度。將原始數據自讀數器(Gen5 2.0軟體)輸出為excel格式且在excel中進一步分析。使用GraphPad Prism 6軟體及邏輯4PL回歸模型生成標準曲線。 表14：來自使用未偶聯寡核苷酸及GalNAc偶聯之寡核苷酸治療之聚(I:C)小鼠(n=3)之總肝懸浮液、肝細胞及非實質細胞中的PD-L1表現及寡核苷酸含量。 細胞類型 CMP ID no PD-L1 表現 ( 鹽水- 聚(I:C) 之%) 寡核苷酸含量 (ng/105 個細胞 ) Avg SD Avg SD 總肝臟細胞 748_1 31 12.4 2.3 0.3 759_2 28 5.3 8.3 1.1 肝細胞 748_1 33 8.0 5.1 3.7 759_2 7 1.0 43.8 18.9 非實質細胞 748_1 31 10.1 2.2 0.7 759_2 66 1.6 1.7 0.9 結果展示，裸(CMP ID NO: 748_1)及偶聯(CMP ID NO: 759_2)寡核苷酸同等程度地降低總肝臟細胞中之PD-L1 mRNA。在經分離肝細胞中，偶聯寡核苷酸之效應幾乎強於裸寡核苷酸之效應5倍，而裸寡核苷酸展示其效應強於非實質細胞中之GalNAc偶聯之寡核苷酸兩倍。在肝細胞及非實質細胞中， PD-L1 mRNA表現之降低在一定程度上與該等細胞類型中之寡核苷酸含量相關。 實例5 -使用裸及GalNAc偶聯之PD-L1反義寡核苷酸之AAV/HBV小鼠中之活體內PD-L1敲低  在本發明研究中，使用裸或GalNAc偶聯之PD-L1反義寡核苷酸治療AAV/HBV小鼠，且在肝中評估PD-L1 mRNA表現及HBV基因表現。 在第-1週使用媒劑(鹽水)、裸PD-L1反義寡核苷酸(CMP ID NO 752_1，在5 mg/kg下，經皮下)及GalNAc PD-L1反義寡核苷酸(CMP ID NO 763_2，在7 mg/kg下，經皮下)預處理5-8週齡雌性HLA-A2/DR1小鼠(5隻動物/組)，該等劑量對應於等莫耳濃度之寡核苷酸。在第0週藉由5× 1010 vg AAV-HBV轉導小鼠(關於其他細節，參見材料及方法部分中之AAV/HBV小鼠模型闡述)。自AAV-HBV轉導後W1至W4，使小鼠接受4次PD-L1寡核苷酸或媒劑(鹽水溶液)之其他皮下注射且相隔一週給予。 在轉導之前一週及在每一注射之後一週獲取血樣。 在最後注射之後兩週將小鼠處死且在PBS灌注後取出其肝。將肝切割成較小切片且直接冷凍。 為量測HBV基因表現，利用Qiagen Biorobot使用 the QIAamp One for all核酸套組(目錄編號965672)自血清提取DNA，將血清以1:20稀釋度稀釋於PBS中，在200ul緩衝液AL中裂解總共100 µl。自套組在100 µl中洗脫DNA。 對於實時qPCR而言，將TaqMan Gene Expression Master Mix (目錄編號4369016，Applied Biosystems)與藉由添加1:1:0.5之下列引子F3_core、R3_core、P3_core (Integrated DNA Technologies，皆各自以100uM重構)來製得之引子混合物一起使用： 正向(F3_core)：CTG TGC CTT GGG TGG CTT T (SEQ ID NO: 784) 反向(R3_core)：AAG GAA AGA AGT CAG AAG GCA AAA(SEQ ID NO: 785) 探針(P3_core)：56-FAM-AGC TCC AAA/ZEN/TTC TTT ATA AGG GTC GAT GTC CAT G-3IABkFQ(SEQ ID NO: 786) 使用10倍稀釋液(始於1×109 個拷貝/µl至1個拷貝/µl且以5µl/反應使用)製得使用HBV質體(基因型D，GTD)之標準曲線。 對於每一反應而言，添加10µl Gene Expression Master Mix、4.5µl水、0.5µl引子混合物及5µl試樣或標準且運行qPCR。 對於分析而言，使用標準曲線計算拷貝數/ml/孔。結果展示於表15中。 使用qPCR量測PD-L1 mRNA表現。 自添加至含有陶瓷珠粒(Lysing Matrix D管，116913500, mpbio)及1ml Trizol之2ml管中之經冷凍肝切片提取mRNA。 使用Precellys組織破碎器將肝切片均質化。將200µl氯仿添加至均質物中，渦旋且在4℃及10000rpm下離心20min。將含有RNA之澄清相(大約500ul)轉移至新管中且添加相同體積之70% EtOH。在充分混合之後，將溶液轉移至RNeasy自旋管柱上且遵循RNeasy套組之人工RNeasy微型套組(目錄編號74104，Qiagen) (包含RNA消解RNase-free DNase Set，目錄編號79254) 進一步提取RNA。在50µl H2 O中洗脫。對於所有試樣而言，量測最終RNA濃度且調節至100ng/ul。 根據製造商說明書在7.5µl RNA下使用Taqman RNA-to-ct 1-step套組(目錄編號4392938，Thermo Fisher)實施qPCR。所用引物混合物含有PD-L1_1-3 (引物編號Mm00452054_m1、Mm03048247_m1及Mm03048248_m1)及內源性對照(ATCB Mm00607939_s1、CANX Mm00500330_m1、YWHAZ Mm03950126_s1及GUSB Mm01197698_m1) 使用2^-ddct方法分析數據。使用所有4種內源性對照之平均值來計算dct值。PD-L1表現係相對於內源性對照之平均值且以鹽水之%形式 表15：使用未偶聯及GalNAc偶聯之寡核苷酸治療之AAV/HBV小鼠(n=5)中之PD-L1 mRNA表現及HBV DNA。 CMP ID no PD-L1 mRNA 表現 ( 鹽水之%) HBV DNA 表現 ( 鹽水之 %) Avg SD Avg SD 裸 752_1 55 35 72 16 GalNAc偶聯 763_2 34 3 79 9 自該等結果可看到，裸寡核苷酸及GalNAc偶聯之寡核苷酸能夠降低AAV/HBV小鼠之肝中之PD-L1 mRNA表現，其中GalNAc偶聯之寡核苷酸略微較佳。兩種寡核苷酸亦使得血清中之HBV DNA略有降低。 實例6 -對AAV/HBV小鼠中之T細胞反應之活體內效應   在本發明研究中，使用靶向PD-L1之抗體或反義寡核苷酸治療來自巴斯德之AAV/HBV小鼠。反義寡核苷酸係裸反義寡核苷酸或偶聯至GalNAc。在治療期間，使用針對HBs及HBc抗原之DNA疫苗對動物實施免疫(參見材料及方法部分)以確保T細胞由抗原呈遞細胞有效引發。評估該治療如何影響肝及脾中之細胞群體以及該等群體上之PD-L1表現及是否可鑑別HBV特異性T細胞反應。治療方案 ： 根據下文方案來治療雌性HLA-A2/DR1小鼠。在兩個單獨子研究中實施研究，其中投與方案略有差異，如下表16及17中所指示。 如材料及方法部分中所闡述來投與DNA疫苗及抗PD-L1抗體。所用反義寡核苷酸係5 mg/kg CMP ID NO 748_1 (裸)及7mg/kg CMP ID NO: 759_2 (GalNAc偶聯)，二者皆係以皮下注射(s.c.)形式來投與。 表16：使用DNA疫苗及DNA疫苗+抗PD-L1抗體之AAV/HBV小鼠治療方案，每組6隻小鼠 天 媒劑 (第10組) DNA疫苗 (第11組) DNA疫苗+抗PDL-1 Ab (第13組) 0 AAV/HBV 29* 動物隨機化 34 鹽水+同型 - Ab 41 鹽水+同型 - Ab 48 鹽水+同型 - Ab 50 - CaTx CaTx 55* PBS+同型 DNA DNA+Ab 62 鹽水+同型 - Ab 69 PBS+同型 DNA DNA+Ab 76* 鹽水+同型 - Ab 83 鹽水+同型 - Ab 97* 處死 同型=小鼠IgG對照Ab，CaTx =心臟毒素，DNA = DNA疫苗，Ab=抗PD-L1 Ab且*=血清收集 表17：使用DNA疫苗及DNA疫苗+裸或偶聯PD-L1寡核苷酸(ASO)之AAV/HBV小鼠治療方案，每組7隻小鼠 天 媒劑 (第1組) DNA疫苗 (第2組) DNA疫苗 + PDL-1 ASO (第7組) DNA疫苗 + GN-PDL-1 ASO (第8組) 0 AAV/HBV 29* 動物隨機化 39 鹽水 鹽水       41    鹽水 ASO GN-ASO 46 鹽水 鹽水       49    鹽水 ASO GN-ASO 53 鹽水 鹽水       55 CaTx CaTx CaTx CaTx 56    鹽水 ASO GN-ASO 59 PBS+鹽水 DNA+PBS DNA DNA 62*    鹽水 ASO GN-ASO 67 鹽水 鹽水       70    鹽水 ASO GN-ASO 74 PBS+鹽水 DNA+PBS DNA DNA 77    鹽水 ASO GN-ASO 81 鹽水 鹽水       84*    鹽水 ASO GN-ASO 88 鹽水 鹽水       91    鹽水 ASO GN-ASO 102 處死 DNA = DNA疫苗，CaTx =心臟毒素，Ab=抗PD-L1 Ab，ASO=裸PDL-1寡核苷酸，GN-ASO= GalNAc-PDL-1寡核苷酸且*=血清收集 在處死時，收集來自每一組之每一小鼠之血液、脾及肝單核細胞且消耗紅血球(裂解緩衝液，BD biosciences, 555899)。肝單核細胞需要如材料及方法部分中所闡述之特定製備。細胞群體 ： 在肝中，藉由肝單核細胞上之表面標記(參見材料及方法)使用細胞術來分析細胞群體。 與對照組(亦即媒劑及DNA免疫化組)相比，經治療小鼠之脾及肝中之NK細胞頻率並未發現顯著變化。表18展示，在肝中，使用裸PD-L1寡核苷酸(CMP ID NO 748_1)及GalNAc偶聯之PD-L1寡核苷酸(CMP ID NO: 759_2)治療之組之T細胞數與亦呈現於圖10 A中的任一對照組(亦即媒劑及DNA免疫化組)相比顯著增加。此增加係源於CD4+及CD8+ T細胞群體有所增加(分別在表18及圖10B及10C中)。 表18：在治療後之肝中之T細胞(以百萬個細胞之形式)    T細胞 (百萬) CD4+ T細胞 (百萬) CD8+ T細胞 (百萬) Avg Std Avg Std Avg Std 媒劑(組1) 0.77 0.44 0.51 0.35 0.11 0.05 DNA疫苗(組2) 0.90 0.24 0.58 0.16 0.16 0.08 DNA疫苗+ 抗PD-L1 Ab (組13) 1.98 0.90 1.40 0.81 0.41 0.23 媒劑(組10) 1.73 0.87 1.13 0.55 0.40 0.25 DNA疫苗(組11) 1.27 0.97 0.79 0.58 0.32 0.32 DNA疫苗+ PD-L1 ASO(組7) 3.78 1.31 2.46 0.72 0.79 0.39 DNA疫苗+ GN-PD-L1 ASO(組8) 3.33 0.66 2.18 0.40 0.67 0.17 PD-L1 表現： 在處死時，在來自脾及肝之巨噬球、B及T細胞上評估PD-L1蛋白之表現。PD-L1抗體在表面標記抗體混合物(參見材料及方法)中之存在使得可藉由細胞術量化PD-L1表現細胞。 在脾中，據觀察，在治療之間表現PD-L1之巨噬球、B細胞及CD4+ T細胞之%並無顯著差異。使用裸PD-L1寡核苷酸(CMP ID NO 748_1)及GalNAc偶聯之PD-L1寡核苷酸(CMP ID NO: 759_2)治療之小鼠中之表現PD-L1之CD8+ T細胞的%低於其他治療(數據未展示)。 在肝中，PD-L1主要以32%之平均頻率表現於CD8+ T細胞上且在對照組(分別係兩個媒劑及DNA接種疫苗組組合，圖11A)中為41%。使用裸PD-L1寡核苷酸或GalNAc PD-L1寡核苷酸進行治療使得表現PD-L1之CD8+ T細胞之頻率有所降低(參見表19及圖11A)。亦在ASO治療之後針對B細胞及CD4+ T細胞觀察到表現PD-L1之細胞%具有顯著差異，但該等細胞類型表現顯著小於CD8+ T細胞之PD-L1 (參見表19及圖11B及C)。使用抗PD-L1 Ab進行治療亦使得所有細胞類型中之PD-L1表現明顯降低。然而，此降低可能係由於PD-L1表位由用於治療之抗PD-L1抗體部分地阻斷，從而防止表面標記抗體混合物中之PD-L1檢測抗體預防結合至PD-L1。因此，藉由用於治療之抗PD-L1抗體所達成之PD-L1下調似乎可為治療抗體與檢測抗體之間之表位競爭的結果。 表19：具有PD-L1表現之肝臟細胞群體之%    CD8+ T細胞之% CD4+ T細胞之% B細胞之%    Avg Std Avg Std Avg Std 媒劑(組10) 35.5 4.7 0.75 0.52 5.9 1.5 DNA疫苗 (第11組) 36.8 7.7 0.61 0.08 5.5 1.1 DNA疫苗+ 抗PD-L1 Ab (第13組) 18.6 12.3 0.33 0.10 2.9 1.7 媒劑 (第1組) 28.5 11.5 0.64 0.21 5.9 1.7 DNA疫苗 (第2組) 44.9 14.4 1.43 0.69 8.7 3.1 DNA疫苗+ PD-L1 ASO (第7組) 9.6 2.4 0.37 0.21 2.9 0.8 DNA疫苗+ GN-PD-L1 ASO (第8組) 14.6 3.3 0.31 0.11 2.8 0.8 HBV 特異性 T 細胞反應： 使用檢測IFNγ及TNFα產生之細胞內細胞介素染色分析(參見材料及方法部分)檢測產生促發炎性細胞介素之NK細胞及CD4+及CD8+ T細胞。 在脾中，在處死時，未檢測到分泌IFNγ-及TNFα之NK細胞且可檢測到少量CD4+ T細胞(頻率＜ 0.1%)。在使用裸PD-L1寡核苷酸或GalNAc PD-L1寡核苷酸治療之小鼠以及此研究中僅接受DNA疫苗之小鼠中檢測靶向兩種HBV抗原之產生IFNγ的CD8+ T細胞(數據未展示)。 在DNA免疫化HBV攜載小鼠之肝中，在處死時並未檢測到產生IFNγ之NK細胞，而在少數DNA免疫化小鼠之肝中以低頻率檢測到對核心或S2+S具有特異性之產生IFNγ之CD4+ T細胞(＜ 0.4%，數據未展示)。在大部分DNA免疫化小鼠中檢測到產生IFNγ之HBV S2+S 特異性CD8+ T細胞。在使用DNA疫苗及裸PD-L1寡核苷酸或GalNAc PD-L1寡核苷酸之組合治療之小鼠中，分泌IFNγ之CD8+ T細胞之頻率有所增加，而使用抗PD-L1抗體之治療並不向DNA接種疫苗增加任何明顯額外效應(圖12)。在大部分DNA免疫化組(除抗PD-L1抗體外)中檢測到靶向套膜蛋白及核心抗原之產生IFNγ之CD8+ T細胞(圖12B)。大部分S2-S特異性T細胞產生IFNγ及TNFα (圖12C)。結果亦展示於表20中。 表20：來自總IFNγ或IFNγ + TNFα細胞群體之HBV抗原(S2-S或核心)特異性CD8+ T細胞之%    PreS2-S特異性T細胞 (IFNγ細胞之%) 核心特異性T細胞 (IFNγ細胞之%) S2-S特異性T細胞 (IFNγ + TNFα之%)    Avg Std Avg Std Avg Std 媒劑 (第10組) 0.15 0.37 0.18 0.43 0.00 0.00 DNA疫苗 (第11組) 1.48 1.10 0.47 0.53 0.42 1.02 DNA疫苗+ 抗PDL-1 Ab 1.18 0.95 0 0 0.38 0.49 媒劑 (第1組) 0.17 0.45 0.11 0.28 0.00 0.00 DNA疫苗 (第2組) 1.70 1.02 0.27 0.51 0.98 0.90 DNA疫苗+ PDL-1 ASO 2.56 1.60 0.78 0.80 1.44 1.55 DNA疫苗+ GN-PDL-1 ASO 3.83 2.18 0.68 1.16 2.62 1.62 實例7 -對AAV/HBV小鼠血清中之HBV抗原及HBV DNA之活體內效應  在本發明研究中，使用GalNAc偶聯之PD-L1反義寡核苷酸CMP ID NO 759_2治療來自上海之AAV/HBV小鼠(參見材料及方法部分)。 評估該治療與媒劑治療動物相比如何影響血清中之HBe及HBs抗原及HBV DNA含量。治療方案 ： 在此研究中使用如在材料及方法部分中之上海模型下所闡述感染攜載HBV基因體(AAV/HBV)之重組腺相關病毒(AAV)之雄性C57BL/6小鼠。經8週每週一次向小鼠(6小鼠/組)注射5 mg/kg反義寡核苷酸CMP ID NO: 759_2或媒劑(鹽水)，其中二者皆係如皮下注射(s.c.)所投與。在治療期間每週以及在治療後6週收集血樣。如下文所闡述在血清試樣中量測HBV DNA、HBsAg及HBeAg含量。前10週之結果展示於表21及圖13中。在歸檔時研究仍在進行，因此，剩餘4週之數據尚未獲得。HBsAg 及 HBeAg 檢測： 在經感染AAV-HBV小鼠中使用HBsAg化學發光免疫分析(CLIA)及HBeAg CLIA套組(Autobio diagnostics Co. Ltd., Zhengzhou,China，目錄編號分別為CL0310-2及CL0312-2)根據製造商方案在血清中來測定血清HBsAg及HBeAg含量。簡言之，將50 μl血清轉移至各別抗體塗覆微量滴定板中且添加50 μl酶偶聯物試劑。將板在室溫下於振盪器上培育60 min，然後利用洗滌緩衝液使用自動洗滌器將所有孔洗滌6次。向每一孔中添加25 μl受質A且然後添加25 μl受質B。將板在室溫下培育10 min，然後使用Envision發光讀數儀量測發光。以單位IU/ml給出HBsAg；其中1 ng HBsAg =1.14 IU。以單位NCU/ml血清給出HBeAg。HBV DNA 提取及 qPCR ： 首先，使用磷酸鹽緩衝鹽水(PBS)將小鼠血清稀釋10倍(1:10)。使用MagNA Pure 96 (Roche)機器人提取DNA。將50μl經稀釋血清在處理柱中與200ul MagNA Pure 96外部裂解緩衝液(Roche，目錄編號06374913001)一起混合且培育10分鐘。然後使用「MagNA Pure 96 DNA及Viral Nucleic Acid Small Volume Kit」 (Roche，目錄編號06543588001)及「Viral NA Plasma SV external lysis 2.0」方案提取DNA。DNA洗脫體積為50 μl。 使用Taqman qPCR機器(ViiA7, life technologies)來量化所提取HBV DNA。一式兩份在PCR中測試每一DNA試樣。將5μl DNA試樣添加至384孔板中含有10 μl TaqMan Gene Expression Master Mix (Applied Biosystems，目錄編號4369016)、0.5 μl PrimeTime XL qPCR引子/探針(IDT)及4.5μl蒸餾水之15μl PCR標準混合物中且使用下列設置實施PCR：UDG培育(2min, 50℃)，酶活化(10min, 95℃)及PCR (40個循環，其中在95℃下變性15sec且在60℃下退火及延伸1min)。自Ct 值基於HBV質體DNA標準曲線藉由ViiA7軟體來計算DNA拷貝數。 用於TaqMan引子及探針(IDT)之序列： 正向核心引子(F3_core)：CTG TGC CTT GGG TGG CTT T(SEQ ID NO: 784) 反向引子(R3_core)：AAG GAA AGA AGT CAG AAG GCA AAA (SEQ ID NO: 785) Taqman探針(P3_core)：56-FAM/AGC TCC AAA /ZEN/TTC TTT ATA AGG GTC GAT GTC CAT G/3IABkFQ (SEQ ID NO: 786)。 表21：在使用GalNAc偶聯之PD-L1反義寡核苷酸治療後來自AAV/HBV小鼠之血中之清HBV-DNA、HBsAg及HBeAg含量。 鹽水 CMP ID NO: 759_2 ， 在5 mg/kg 下 HBV-DNA HBsAg HBeAg HBV-DNA HBsAg HBeAg 天數 Avg Std Avg Std Avg Std Avg Std Avg Std Avg Std 0 7.46 0.35 3.96 0.48 3.23 0.14 7.44 0.29 3.87 0.40 3.17 0.13 7 7.53 0.23 4.17 0.45 3.35 0.10 7.53 0.20 3.91 0.42 3.19 0.18 14 7.57 0.24 4.12 0.49 3.19 0.11 7.45 0.22 3.90 0.50 2.99 0.27 21 7.47 0.27 3.93 0.51 3.12 0.05 7.33 0.47 3.71 0.76 2.78 0.26 28 7.68 0.26 3.88 0.67 3.18 0.13 7.45 0.46 3.65 0.93 2.67 0.38 35 7.69 0.21 4.03 0.54 2.95 0.08 7.13 0.75 2.98 1.05 2.04 0.38 42 7.58 0.23 3.89 0.65 3.34 0.10 6.69 0.89 2.60 1.05 1.98 0.45 49 7.77 0.17 3.54 1.06 3.08 0.26 6.56 1.26 2.19 0.70 1.47 0.37 56 7.71 0.24 3.99 0.86 3.28 0.05 6.21 1.48 2.28 0.84 1.38 0.30 63 7.59 0.28 3.67 1.07 3.25 0.13 6.08 1.39 2.08 0.71 1.35 0.30 自此研究可看到，在治療6週之後，GalNAc偶聯之PD-L1反義寡核苷酸CMP NO 759_2對血清中之HBV-DNAH、BsAg及HBeAg含量降低具有顯著效應，且在治療之後持續至少2週之效應已終止。 實例8 -使用GalNAc偶聯之PD-L1寡核苷酸之人類原代肝細胞中之活體外PD-L1敲低  使用基因體學探究GalNAc偶聯之PD-L1反義寡核苷酸化合物降低原代人類肝細胞中之PD-L1轉錄之能力。細胞培養物 將經冷藏保存之人類肝細胞以大約5 × 106 個細胞/ml之密度懸浮於補充有10%胎牛血清、青黴素(100 U/ml)、鏈黴素(0.1 mg/ml)及L-麩醯胺酸(0.292 mg/ml)之WME中且以2 × 105 細胞/孔之密度接種至膠原塗覆之24孔板中(Becton Dickinson AG, Allschwil, Switzerland)。在開始使用寡核苷酸以100 µM之最終濃度進行處理之前，將細胞預培養4h以使得連接至細胞培養板。所用寡核苷酸展示於表21及表8中，媒劑係PBS。將接種培養基更換為315 µl無血清WME (補充有青黴素(100 U/ml)、鏈黴素(0.1 mg/ml)、L-麩醯胺酸(0.292 mg/ml))且將35 µl於PBS中之1 mM寡核苷酸儲備溶液添加至細胞培養物中並在細胞上保留24小時或66小時。文庫製備 使用Illumina Stranded mRNA化學在Illumina測序平臺上利用2 × 51 bp配對端讀數及每一樣品中30M之最小讀取深度之測序策略(Q squared EA)來描述轉錄物表現。藉由添加350 µl Qiagen RLT緩衝液來在孔中裂解細胞且登錄於隨機化方案中。 使用Qiagen RNeasy微型套組純化mRNA。量化mRNA且使用Agilent生物分析儀評價完整性。在經分離RNA之初始品質評價後，據觀察，所有試樣皆符合100ng之輸入品質度量且RIN評分＞7.0。 使用Illumina TruSeq Stranded mRNA文庫製備自100 ng總RNA開始來生成用於所有試樣之測序文庫。使用Agilent生物分析儀(DNA 1000套組)分析最終cDNA文庫之大小分佈，藉由qPCR (KAPA Library Quant套組)量化且在製備中正規化至2 nM以用於測序。使用標準簇生成套組v5來使cDNA文庫以等溫方式結合至流動槽表面及cBot以將連接cDNA構築體各自擴增至最高約1000個拷貝之純系簇。藉由邊合成邊測序技術使用TruSeq SBS套組測定DNA序列。數據處理 使用GSNAP短讀數比對程式將長2×51 bp之Illumina配對端測序讀數定位於人類參考基因體hg19上。使用SAMTOOLS程式將SAM格式比對轉換成分選比對BAM格式檔案。基於來自NCBI RefSeq之外顯子注釋(由hg19之相應GTF檔案指定)來估計PD-L1之基因讀取計數。使用DESeq2 R套件應用考慮每一試樣之不同文庫大小之正規化步驟。 在與GalNAc偶聯之PD-L1反義寡核苷酸化合物一起培育之後之PD-L1轉錄降低展示於表22中。 表22：在使用GalNAc偶聯之寡核苷酸處理後原代一級肝細胞中之PD-L1轉錄降低，n=4 化合物 PD-L1 表現程度 ，24 h ( 文庫大小調節之計數) PD-L1 表現程度 ，66 h ( 文庫大小調節之計數) 媒劑 259 156 159 168 192 136 202 211 767_2 7 7 11 14 22 9 28 15 766_2 16 13 15 10 17 11 29 13 769_2 15 21 18 18 25 18 26 25 768_2 41 25 27 48 31 25 34 22 770_2 21 16 44 62 67 51 38 63 在培育24及66小時之後，在與使用媒劑處理之試樣相比時，所有5種GalNAc偶聯之反義化合物皆展示顯著PD-L1轉錄降低。 實例9 -偶聯及裸PD-L1反義寡核苷酸在HBV感染之ASGPR-HepaRG細胞中之EC50  在HBV感染之ASGPR-HepaRG細胞中比較兩種裸及等效GalNAc偶聯PD-L1反義寡核苷酸之功效。細胞系 將HepaRG細胞(Biopredic International, Saint-Gregoire, France)培養於威廉E培養基(補充有10% HepaRG生長補充物(Biopredic))中。自此細胞系，使用慢病毒方法生成穩定過度表現人類ASGPR1及ASGPR2之HepaRG細胞系。以MOI 300使用由Sirion biotech按需產生之編碼人類ASGPR1及2之慢病毒(CLV-CMV-ASGPR1-T2a_ASGPR2-IRES-Puro)在CMV啟動子及嘌呤黴素抗性基因之控制下來轉導增殖HepaRG細胞。使用1µg/ml嘌呤黴素經11天選擇轉導細胞且然後維持於相同濃度之抗生素中以確保轉基因之穩定表現。在mRNA層面下藉由RT-qPCR (ASGPR1：8560倍對非轉導，ASGPR2：2389倍對非轉導)及在蛋白質層面下藉由流式細胞術分析來證實ASGPR1/2過度表現。 在感染之前使用1.8% DMSO分化細胞至少2週。HBV基因型D係衍生自HepG2.2.15細胞培養上清液且使用PEG沈澱濃縮。為評估測試化合物針對HBV之活性，使用HBV以20至30之MOI感染96孔板中之分化ASGPR-HepaRG細胞20 h，然後使用PBS將細胞洗滌4次以去除HBV接種物。寡核苷酸功效 將下列寡核苷酸 裸PD-L1 ASO 等效GalNAc 偶聯PD-L1 ASO CPM ID NO: 640_1 CPM ID NO: 768_2 CPM ID NO: 466_1 CPM ID NO: 769_2 在感染後第7天及第10天使用25µM至0.4 nM之連續稀釋液(於PBS中以1:4稀釋)添加至HBV感染之ASGPR-HepaRG細胞中。在感染後第13天收穫細胞。 根據製造商說明書使用MagNA Pure 96 Cellular RNA大體積套組在MagNA Pure 96系統(Roche Diagnostics)上來提取總mRNA。對於基因表現分析而言，如實例5中所闡述來實施RT-qPCR。 使用2^-ddct方法分析數據。使用肌動蛋白B作為內源性對照以計算dct值。PD-L1表現係相對於內源性對照及鹽水媒劑。 在GraphPad Prism6中實施EC50計算且展示於表23中。 表23：ASGPR-HepaRG HBV感染細胞中之EC50，n=4。 CMP ID NO EC50 (µM) 640_1 2.25 768_2 0.10 466_1 5.82 769_2 0.13 該等數據明確展示，PD-L1反義寡核苷酸之GalNAc偶聯物顯著改良EC50值。 實例10 -衍生自慢性HBV患者之PBMC中之T細胞功能刺激  探究在末梢血單核細胞(PBMC)之離體HBV抗原刺激之後裸PD-L1反義化合物是否可增加慢性感染HBV (CHB)患者之T細胞功能。 將來自三名慢性HBV感染患者之冷凍PBMC解凍且以200’000個細胞/孔之密度接種於100µl培養基(RPMI1640 + GlutaMax+ 8%人類血清+ 25mM Hepes + 1% PenStrep)中。第二天，使用1µM PepMix HBV大套膜蛋白或1µM PepMix HBV核心蛋白(參見表9)使用或不使用5µM CMP ID NO: 466_1或CMP ID NO: 640_1在100µl含有100pg/ml IL-12及5ng/ml IL-7之培養基中刺激細胞(在第8天僅施加伴刀豆球蛋白(Concanavalin)刺激)。4天後，使用含有50IU IL-2之培養基更新PD-L1反義寡核苷酸處理。在第一刺激之後第8天，使用PepMix或5µg/ml伴刀豆球蛋白A以及PD-L1反義寡核苷酸再刺激細胞24h。對於最後5h刺激而言，添加0.1 µl佈雷菲德菌素A (佈雷菲德菌素A)、0.1µl莫能菌素(monensin)及3µl抗人類CD-107 (APC)。 在24h之後，使用染色緩衝液(PBS + 1% BSA + 0.09%疊氮化鈉+ EDTA)洗滌細胞且在4℃下施加表面染色30min [抗人類CD3 (BV 605)、抗人類CD4 (FITC)、抗人類CD8 (BV711)、抗人類PDL1 (BV421)、抗人類PD1 (PerCP-Cy5.5)及活及死亡染色(BV510) (BD Biosciences)]。將細胞在4℃下固定於BD固定緩衝液中保持15min。次日早上，使用BD Perm/Wash緩衝液將細胞在4℃下滲透15min且在4℃下實施細胞內染色30mi [抗人類INF( (PE)]。在Perm/Wash緩衝液中洗滌之後，將細胞溶於250µl染色緩衝液中。 在BD Fortessa (BD Biosciences)上實施FACS量測。對於分析而言，首先將整個細胞群體選通於活細胞上(活及死亡染色，BV510)，且然後選通於CD3+ (BV605)細胞上。然後將CD3+細胞繪圖為CD107a+ (APC)對IFNγ+ (PE)。 結果展示於表24中。 表24：PD-L1 ASO處理對來自分離自三名慢性HBV感染患者之PBMC之CD3+ T細胞之效應。    無抗原刺激 套膜蛋白抗原 核心抗原 鹽水 CMP 466_1 CMP 640_1 鹽水 CMP 466_1 CMP 640_1 鹽水 CMP 466_1 CMP 640_1 INFγ-/ CD107+ 1.16 4.95 4.81 4.7 9.12 8.62 3.84 9.66 7.31 2.7 3.59 2.74 2.57 3.69 3.2 3.25 3.34 2.92 3 3.87 3.98 4.59 12.5 10.9 9.23 6.11 6.88 INFγ+/ CD107+ 0.12 1.03 1.15 3.19 17.3 18.9 2.38 15.1 5.75 0.49 3.12 1.75 2.73 7 5.34 1.63 2.35 1.9 0.24 1.13 1.5 1.6 8.16 3.06 1.68 1.9 1.91 INFγ+/ CD107- 0.33 1.43 1.08 5.11 7.74 9.47 3.14 7.76 2.83 0.61 2.9 2.26 7.84 5.79 5.78 2.33 2.82 2.95 0.17 1.57 1.72 1.22 2.58 0.99 0.1 0.61 1.04 自該等數據可看到，抗原刺激本身能夠誘導CHB患者(n=3)之PBMC中之T細胞活化(增加表現INFγ(及/或CD107a之CD3+細胞之%)。添加PD-L1反義寡核苷酸CMP 466_1或640_1使得額外增加CD3+ T細胞反應。此增加主要於HBV套膜蛋白刺激組中觀察到。definitionOligonucleotides As generally understood by those familiar with the art, the term "oligonucleotide" as used herein is defined as a molecule that includes two or more covalently linked nucleosides. These covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually produced in the laboratory by solid-phase chemical synthesis and subsequent purification. When referring to the sequence of an oligonucleotide, one may refer to a covalently linked nucleotide or nucleobase portion of a nucleoside or a modified sequence or sequence thereof. The oligonucleotides of the present invention are artificially prepared and synthesized chemically, and are usually purified or isolated. The oligonucleotides of the invention may include one or more modified nucleosides or nucleotides.Antisense oligonucleotide The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide capable of modulating the expression of a target gene by hybridizing to a target nucleic acid, especially an adjacent sequence on the target nucleic acid. Antisense oligonucleotides are not double-stranded in nature and therefore are not siRNAs. Preferably, the antisense oligonucleotides of the present invention are single-stranded.Adjacent nucleotide sequence The term "contiguous nucleotide sequence" refers to the region of the oligonucleotide that is complementary to the target nucleic acid. The term can be used interchangeably herein with the term "adjacent nucleobase sequence" and the term "oligonucleotide motif sequence". In some embodiments, all nucleotides of the oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, the oligonucleotide includes a contiguous nucleotide sequence and optionally other nucleotides (e.g., a nucleotide linker region that can be used to connect a functional group to the contiguous nucleotide sequence). The nucleotide linker region may or may not be complementary to the target nucleic acid.Nucleotide Nucleotides are the building blocks of oligonucleotides and polynucleotides and for the purpose of the present invention they include natural and non-natural nucleotides. In nature, nucleotides (such as DNA and RNA nucleotides) include ribose sugar moieties, nucleobase moieties, and one or more phosphate groups (which are not present in nucleosides). Nucleosides and nucleotides can also be referred to interchangeably as "units" or "monomers."Modified nucleoside The term "modified nucleoside" or "nucleoside modification" as used herein refers to the modification by introducing one or more sugar moieties or (nucleo) base moieties compared to equivalent DNA or RNA nucleosides. Nucleoside. In a preferred embodiment, the modified nucleoside includes a modified sugar moiety. The term modified nucleoside may also be used interchangeably with the term "nucleoside analog" or modified "unit" or modified "monomer" herein.Modified internucleoside linkage As generally understood by those familiar with the art, the term "modified internucleoside linkage" is defined as a linkage that covalently couples two nucleosides together in addition to a phosphodiester (PO) linkage. Nucleosides with modified internucleoside linkages are also called "modified nucleotides". In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to the phosphodiester linkage. For natural oligonucleotides, internucleoside linkages include phosphate groups that create phosphodiester bonds between adjacent nucleosides. The modified internucleoside linkage is particularly useful for stabilizing oligonucleotides for in vivo applications, and can be used to prevent nuclease cleavage in the DNA or RNA nucleoside region of the oligonucleotides of the present invention (e.g., in gaps). In the gap region of the body oligonucleotide and in the region of the modified nucleoside). In one embodiment, the oligonucleotide includes one or more internucleoside linkages modified from natural phosphodiesters to, for example, linkages that are more resistant to nuclease attack. Nuclease resistance can be determined by culturing oligonucleotides in serum or by using nuclease resistance analysis (such as snake venom phosphodiesterase (SVPD)), both of which are well known in the industry. Internucleoside linkages that can enhance the nuclease resistance of oligonucleotides are called nuclease-resistant internucleoside linkages. In some embodiments, at least 50% of the internucleoside linkages in the modified oligonucleotide or its adjacent nucleotide sequence, for example, at least 60% of the modified oligonucleotide or its adjacent nucleotide sequence , For example at least 70%, for example at least 80 or for example at least 90% of the internucleoside linkages. In some embodiments, all internucleoside linkages of the modified oligonucleotide or its adjacent nucleotide sequence are modified. It should be appreciated that, in some embodiments, the nucleoside linking the oligonucleotide of the invention to a non-nucleotide functional group (eg, a conjugate) may be a phosphodiester. In some embodiments, all internucleoside linkages of the oligonucleotide or its adjacent nucleotide sequence are nuclease resistant internucleoside linkages. The modified internucleoside linkage may be selected from the group including phosphorothioate, phosphorodithioate, and borane phosphate. In some embodiments, the modified internucleoside linkage is compatible with RNaseH recruitment of the oligonucleotides of the invention, such as phosphorothioate, phosphorodithioate, or borane phosphate. In some embodiments, the internucleoside linkage includes sulfur (S), such as phosphorothioate internucleoside linkage. Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics, and ease of manufacture. In some embodiments, at least 50% of the internucleoside linkages in the oligonucleotide or its adjacent nucleotide sequence are phosphorothioate, for example, in the oligonucleotide or its adjacent nucleotide sequence At least 60%, such as at least 70%, such as at least 80 or such as at least 90% of the internucleoside linkage is phosphorothioate. In some embodiments, all internucleoside linkages of the oligonucleotide or its adjacent nucleotide sequence are phosphorothioates. In some embodiments, the oligonucleotide includes one or more neutral internucleoside linkages, especially selected from the group consisting of phosphotriester, methylphosphonate, MMI, amide-3, methylal or thiomethyl The internucleoside linkage of acetals. Other internucleoside linkages are disclosed in WO2009/124238 (incorporated herein by reference). In one embodiment, the internucleoside linkage is selected from the linkers disclosed in WO2007/031091 (incorporated herein by reference). Specifically, the internucleoside linkage can be selected from -O-P(O)2 -O-, -O-P(O,S)-O-, -O-P(S)2 -O-, -S-P(O)2 -O-, -S-P(O,S)-O-, -S-P(S)2 -O-, -O-P(O)2 -S-, -O-P(O,S)-S-, -S-P(O)2 -S-, -O-PO(RH )-O-, O-PO(OCH3 )-O-, -O-PO(NRH )-O-, -O-PO(OCH2 CH2 S-R)-O-, -O-PO(BH3 )-O-, -O-PO(NHRH )-O-, -O-P(O)2 -NRH -, -NRH -P(O)2 -O-, -NRH -CO-O-, -NRH -CO-NRH -, and/or internucleoside linker can be selected from the following groups: -O-CO-O-, -O-CO-NRH -, -NRH -CO-CH2 -, -O-CH2 -CO-NRH -, -O-CH2 -CH2 -NRH -, -CO-NRH -CH2 -, -CH2 -NRH CO-, -O-CH2 -CH2 -S-, -S-CH2 -CH2 -O-, -S-CH2 -CH2 -S-, -CH2 -SO2 -CH2 -, -CH2 -CO-NRH -, -O-CH2 -CH2 -NRH -CO-, -CH2 -NCH3 -O-CH2 -, where RH It is selected from hydrogen and C1-4-alkyl. Nuclease resistant linkages (such as phosphorothioate linkages) are particularly useful in oligonucleotide regions that can recruit nucleases when forming duplexes with target nucleic acids, such as gapmer regions G or head and tail regions. The unmodified nucleoside region of the polymer. However, phosphorothioate linkages can also be used in non-nuclease recruitment regions and/or affinity-enhancing regions (for example, regions F and F'for gapmers or modified nucleoside regions for head and tail polymers) middle. However, each design region may include internucleoside linkages (such as phosphodiester linkages) other than phosphorothioate, especially in regions where modified nucleoside (such as LNA) protective linkages are resistant to nuclease degradation. Incorporating phosphodiester links (such as one or two links, especially between or adjacent to modified nucleoside units (usually located in non-nuclease recruitment regions)) can change the biology of oligonucleotides Availability and/or biodistribution-see WO2008/113832 (incorporated herein by reference). In one embodiment, all internucleoside linkages in the oligonucleotide are phosphorothioate and/or borane phosphate linkages. Preferably, all internucleoside linkages in the oligonucleotide are phosphorothioate linkages.Nucleobase The term nucleobase includes purine (such as adenine and guanine) and pyrimidine (such as uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds in nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases, which may be different from natural nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobases" refer to natural nucleobases (such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine) and unnatural variants. Such variants are, for example, described in Hirao et al. (2012) Accounts of Chemical Research Vol. 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Supplement 37 1.4.1. In some embodiments, the nucleobase moiety is modified by changing purines or pyrimidines into modified purines or pyrimidines (e.g., substituted purines or substituted pyrimidines), for example selected from isocytosine, pseudo-isocytosine, 5-methine Cytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-urine The nucleus of pyrimidine, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine Base. The nucleobase portion can be indicated by the letter code (such as A, T, G, C, or U) used for each corresponding nucleobase, where each letter may optionally include a modified nucleobase with equivalent functions . For example, in the exemplified oligonucleotide, the nucleobase portion is selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.Modified oligonucleotide The term modified oligonucleotide is described as including one or more sugar-modified nucleosides and/or oligonucleotides linked between modified nucleosides. The term "chimeric" oligonucleotide is a term used in the literature to describe oligonucleotides with modified nucleosides.Complementarity The term "complementarity" describes the ability of nucleoside/nucleoside Watson-Crick base pairing. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T)/uracil (U). It should be understood that oligonucleotides may include nucleosides with modified nucleobases, for example, 5-methylcytosine is often used instead of cytosine, and thus the term complementarity encompasses both unmodified nucleobases and modified nucleobases. Watson-Crick base pairing between nucleobases (see, for example, Hirao et al. (2012) Accounts of Chemical Research Volume 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Supplement 37 1.4.1 ). The term "complementary %" as used herein refers to a nucleic acid molecule (e.g., oligonucleotide) that is complementary to the adjacent nucleotide sequence at a given position in a separate nucleic acid molecule (e.g., target nucleic acid) at a given position (i.e. forms Watson -The number of nucleotides (in the form of a percentage of adjacent nucleotide sequences) of the creek base pair). Calculate the percentage by the following method: count the number of aligned bases that form a pair between the two sequences (when aligning the target sequence 5'-3' and 3'-5' oligonucleotide sequences), divide Take the total number of nucleotides in the oligonucleotide and multiply by 100. In this comparison, nucleobases/nucleotides that are not aligned (form a base pair) can be regarded as mismatches. The term "completely complementary" means 100% complementary. The following is an example of an oligonucleotide (SEQ ID NO: 5) that is completely complementary to the target nucleic acid (SEQ ID NO: 772). 5’gcagtagagccaatta3’ (SEQ ID NO:772) 3’cgtcatctcggttaat5’ (SEQ ID NO: 5)consistency As used herein, the term "identity" refers to a nucleic acid molecule (e.g., oligonucleotide) at a given position and a single nucleic acid molecule (e.g., target nucleic acid) at a given position in the adjacent nucleotide sequence identical (that is, capable of complementing Nucleosides form Watson-Crick base pairs) the number of nucleotides (as a percentage of adjacent nucleotide sequences). The percentage is calculated by counting the number of aligned bases that are identical between the two sequences (including gaps), dividing by the total number of nucleotides in the oligonucleotide and multiplying by 100. Percent consistency = (number of matches × 100)/length of comparison area (including gaps).Hybridization The term "hybridizing (hybridizes)" as used herein should be understood as two nucleic acid strands (for example, oligonucleotide and target nucleic acid) forming hydrogen bonds between base pairs on opposite strands, thereby forming a double helix. The binding affinity between two nucleic acid strands is the hybridization strength. It is usually based on the melting temperature (Tm ) To illustrate, the melting temperature is defined as the temperature at which half of the oligonucleotide and the target nucleic acid form a double helix. Under physiological conditions, Tm Is not strictly proportional to affinity (Mergny and Lacroix, 2003,Oligonucleotides 13:515-537). The standard state Gibbs free energy (Gibbs free energy) ΔG° is a more accurate representation of the binding affinity and is related to the dissociation constant of the reaction (Kd ) With ΔG°=-RTln(Kd ) Form correlation, where R is the gas constant and T is the absolute temperature. Therefore, the extremely low ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy related to the reaction, in which the aqueous concentration is 1M, the pH is 7, and the temperature is 37°C. The hybridization of oligonucleotide to target nucleic acid reacts spontaneously and the ΔG° of the spontaneous reaction is less than zero. ΔG° can be measured experimentally by, for example, using the isothermal titration calorimetry (ITC) method, such as Hansen et al., 1965,Chem. Comm. 36-38 and Holdgate et al., 2005,Drug Discov Today As explained in. Those familiar with this technology should know that commercial equipment can be used for ΔG° measurement. It can also be used numerically by using the nearest neighbor model (e.g. by SantaLucia, 1998,Proc Natl Acad Sci USA. 95: 1460-1465) use appropriately derived thermodynamic parameters (by Sugimoto et al., 1995,Biochemistry 34:11211-11216 and McTigue et al., 2004,Biochemistry 43:5388-5405) to estimate ΔG°. In order to adjust its intended nucleic acid target by hybridization, the oligonucleotide of the present invention hybridizes to the target nucleic acid with an estimated ΔG° value of less than -10 kcal (for oligonucleotides of 10-30 nucleotides in length) . In some embodiments, the degree or intensity of hybridization is measured by the standard state Gibbs free energy ΔG°. For oligonucleotides of 8-30 nucleotides in length, the oligonucleotide may be in the range of less than -10 kcal (for example, less than -15 kcal, for example, less than -20 kcal, and for example, less than -25 kcal) The estimated ΔG° value hybridizes to the target nucleic acid. In some embodiments, the oligonucleotide is in the range of (-10 kcal to -60 kcal, such as -12 kcal to -40 kcal, such as -15 kcal to -30 kcal or -16 kcal to -27 kcal, such as -18 kcal The estimated ΔG° value to -25 kcal) hybridizes to the target nucleic acid.Target nucleic acid According to the present invention, the target nucleic acid is a nucleic acid encoding mammalian PD-L1 and can be, for example, a gene, RNA, mRNA and mRNA precursor, mature mRNA or cDNA sequence. The target can thus be referred to as a PD-L1 target nucleic acid. The oligonucleotides of the present invention can, for example, target the exon region of mammalian PD-L1, or can, for example, target the intron region of PD-L1 mRNA precursor (see Table 1). Table 1: Human PD-L1 exons and introns Human PD-L1 mRNA precursor (SEQ ID NO 1) in the exon region Intron region in human PD-L1 mRNA precursor (SEQ ID NO 1) ID Start End ID Start End e1 1 94 i1 95 5597 e2 5598 5663 i2 5664 6576 e3 6577 6918 i3 6919 12331 e4 12332 12736 i4 12737 14996 e5 14,997 15410 i5 15411 16267 e6 16268 16327 i6 16328 17337 e7 17338 20064 Suitably, the target nucleic acid encodes the PD-L1 protein, especially mammalian PD-L1, such as human PD-L1 (see, for example, Tables 2 and 3, which provide reference mRNA and pre-mRNA for human, monkey and mouse PD-L1 Body sequence). In the context of the present invention, the mRNA precursor is also regarded as a nucleic acid encoding a protein. In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO: 1, 2, and 3 or natural variants thereof (for example, a sequence encoding a mammalian PD-L1 protein). If the oligonucleotide of the present invention is used in research or diagnosis, the target nucleic acid can be cDNA or a synthetic nucleic acid derived from DNA or RNA. For in vivo or in vitro applications, the oligonucleotides of the present invention can generally inhibit the expression of the PD-L1 target nucleic acid in cells expressing the PD-L1 target nucleic acid. The contiguous sequence of the nucleobase of the oligonucleotide of the present invention is usually complementary to the PD-L1 target nucleic acid, as measured across the length of the oligonucleotide, with the exception of one or two mismatches as appropriate, and the exclusion may be The oligonucleotide is linked to an optional functional group (e.g. conjugate) or other non-complementary terminal nucleotide based on a nucleotide-based linker region (e.g. region D'or D"). In some embodiments, the target nucleic acid may be RNA or DNA, such as messenger RNA, such as mature mRNA or mRNA precursor. In some embodiments, the target nucleic acid encoding mammalian PD-L1 protein (e.g., human PD-L1) is RNA or DNA (e.g., the human PD-L1 mRNA precursor sequence (e.g., disclosed as SEQ ID NO 1) or has NCBI Human mRNA sequence with reference number NM_014143). Additional information about exemplary target nucleic acids is provided in Tables 2 and 3. Table 2: Genome and assembly information of PD-L1 in each species. Species Chr. chain Genome coordinate start end Combination NCBI Reference Sequence of mRNA *Accession Number Humanity 9 fwd 5450503 5470566 GRCh38:CM000671.2 NM_014143 Crab-eating monkey 15 73560846 73581371 GCF_000364345.1 XM_005581779 Mouse 19 fwd 29367455 29388095 GRCm38:CM001012.2 NM_021893 Fwd = forward chain. Genome coordinates provide mRNA precursor sequences (genome sequences). NCBI reference provides mRNA sequence (cDNA sequence). *National Center for Biotechnology Information reference sequence database is a comprehensive, integrated, non-redundant, fully annotated collection of reference sequences (including genomes, transcripts and proteins). It is deposited at www.ncbi.nlm.nih.gov/refseq. Table 3: Sequence details of PD-L1 in each species. Species RNA type Length (nt) SEQ ID NO Humanity mRNA precursor 20064 1 Crab-eating monkey mRNA precursor GCF ref 20261 2 Crab-eating monkey Pre-mRNA, internal 20340 3 Mouse mRNA precursor 20641 4 Target sequence The term "target sequence" as used herein refers to a nucleotide sequence that includes a nucleobase sequence complementary to the oligonucleotide of the present invention that is present in the target nucleic acid. In some embodiments, the target sequence consists of a region on the target nucleic acid that is complementary to the adjacent nucleotide sequence of the oligonucleotide of the invention. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide, and may, for example, represent a preferred region of the target nucleic acid that can be targeted by several oligonucleotides of the invention. The target sequence may be a subsequence of the target nucleic acid. In some embodiments, the subsequence is a sequence selected from the group consisting of a1-a149 (see Table 4). In some embodiments, the subsequence is selected from human PD-L1 mRNA exons (eg, PD-L1 human mRNA exons selected from the group consisting of: e1, e2, e3, e4, e5, e6, and e7 (See Table 1 above)) The sequence of the group that is composed. In some embodiments, the subsequence is selected from the human PD-L1 mRNA intron (for example, selected from the PD-L1 human mRNA intron group consisting of: i1, i2, i3, i4, i5 and i6 (see above Table 1)) The sequence of the group composed. Oligonucleotides of the present invention include adjacent nucleotide sequences that are complementary to or hybridize to a target nucleic acid (e.g., a subsequence of a target nucleic acid, such as the target sequence described herein). Oligonucleotides include adjacent nucleotide sequences with at least 8 nucleotides that are complementary to or hybridize to a target sequence present in a target nucleic acid molecule. The contiguous nucleotide sequence (and thus the target sequence) includes at least 8 contiguous nucleotides, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides, such as 12-25 contiguous nucleotides, such as 14-18 contiguous nucleotides.Target cell The term "target cell" as used herein refers to a cell that expresses a target nucleic acid. In some embodiments, the target cell may be a target cell in vivo or in vitro. In some embodiments, the target cell line is mammalian cells, such as rodent cells (e.g., mouse cells or rat cells) or primate cells (e.g., monkey cells or human cells). In a preferred embodiment, the target cell expresses PD-L1 mRNA (for example, PD-L1 mRNA precursor or PD-L1 mature mRNA). The polyadenylation tail of PD-L1 mRNA usually ignores antisense oligonucleotide targeting.Natural variant The term "natural variant" refers to a variant of the PD-L1 gene or transcript that is derived from the same locus as the target nucleic acid, but can be, for example, due to the generation of multiple gene codes encoding the same amino acid codons Degeneracy may vary due to alternative splicing of the mRNA precursor or the presence of polymorphism (for example, single nucleotide polymorphism), and refers to allelic variants. Based on the existence of the fully complementary sequence of the oligonucleotide, the oligonucleotide of the present invention can thereby target the target nucleic acid and its natural variants. In some embodiments, the natural variant and the mammalian PD-L1 target nucleic acid (for example, a target nucleic acid selected from the group consisting of SEQ ID NO 1, 2 and 3) have at least 95% (for example at least 98% or at least 99%) The homology. Known many single nucleotide polytypes in the PD-L1 gene, such as those disclosed in the table below (human mRNA precursor start/reference sequence is SEQ ID NO 2) Variation name Variant allele Minor allele Minor allele frequency Starting point on SEQ ID NO: 1 rs73397192 G/A A 0.10 2591 rs12342381 A/G G 0.12 308 rs16923173 G/A A 0.13 14,760 rs2890658 C/A A 0.16 14628 rs2890657 G/C C 0.21 2058 rs3780395 A/G A 0.21 14050 rs147367592 AG/- - 0.21 13425 rs7023227 T/C T 0.22 6048 rs2297137 G/A A 0.23 15230 rs1329946 G/A A 0.23 2910 rs5896124 -/G G 0.23 2420 rs61061063 T/C C 0.23 11709 rs1411263 T/C C 0.23 8601 rs59906468 A/G G 0.23 15583 rs6476976 T/C T 0.24 21012 rs35744625 C/A A 0.24 3557 rs17804441 T/C C 0.24 7231 rs148602745 C/T T 0.25 22548 rs4742099 G/A A 0.25 20311 rs10815228 T/C C 0.25 21877 rs58817806 A/G G 0.26 20769 rs822342 T/C T 0.27 3471 rs10481593 G/A A 0.27 7593 rs822339 A/G A 0.28 2670 rs860290 A/C A 0.28 2696 rs822340 A/G A 0.28 2758 rs822341 T/C T 0.28 2894 rs12002985 C/G C 0.28 6085 rs822338 C/T C 0.28 1055 rs866066 C/T T 0.28 451 rs6651524 A/T T 0.28 8073 rs6415794 A/T A 0.28 8200 rs4143815 G/C C 0.28 17,755 rs111423622 G/A A 0.28 24096 rs6651525 C/A A 0.29 8345 rs4742098 A/G G 0.29 19995 rs10975123 C/T T 0.30 10877 rs2282055 T/G G 0.30 5230 rs4742100 A/C C 0.30 20452 rs60520638 -/TC TC 0.30 9502 rs17742278 T/C C 0.30 6021 rs7048841 T/C T 0.30 10299 rs10815229 T/G G 0.31 22143 rs10122089 C/T C 0.32 13278 rs1970000 C/A C 0.32 14534 rs112071324 AGAGAG/- AGAGAG 0.33 16701 rs2297136 G/A G 0.33 17453 rs10815226 A/T T 0.33 9203 rs10123377 A/G A 0.36 10892 rs10123444 A/G A 0.36 11139 rs7042084 G/T G 0.36 7533 rs10114060 G/A A 0.36 11227 rs7028894 G/A G 0.36 10408 rs4742097 C/T C 0.37 5130 rs1536926 G/T G 0.37 13486 rs1411262 C/T T 0.39 8917 rs7041009 G/A A 0.45 12741 Performance adjustment The term "performance modulation" as used herein is to be understood as a general term regarding the ability of an oligonucleotide to change the amount of PD-L1 (compared to the amount of PD-L1 before the oligonucleotide is administered). Alternatively, a reference control experiment can be used to determine performance adjustment. It is generally understood that a control is an individual or target cell treated or treated with a saline composition or an individual or target cell treated or treated with a non-target oligonucleotide (mock). However, standard care can also be used to treat individuals. A type of regulatory oligonucleotide can, for example, degrade mRNA or block transcription to inhibit, down-regulate, reduce, inhibit, remove, stop, block, prevent, attenuate, reduce, avoid or terminate PD-L1 performance . Another type of regulatory oligonucleotide can restore, increase, or enhance PD-L1 performance, for example, by repairing splicing sites or preventing splicing or removing or blocking inhibitory mechanisms (such as microRNA repression).High affinity modified nucleoside The high-affinity modified nucleoside system, when incorporated into the oligonucleotide, will enhance the affinity of the oligonucleotide for its complementary target (for example, by melting temperature (Tm ) The modified nucleotides measured). The high-affinity modified nucleoside of the present invention preferably increases the melting temperature of each modified nucleoside by +0.5°C to +12°C, more preferably +1.5°C to 10°C, and most preferably +3°C to +8°C . Many high-affinity modified nucleosides are known in the industry and include, for example, many 2'substituted nucleosides and locked nucleic acids (LNA) (see, for example, Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).Sugar modification The oligomers of the present invention may include one or more nucleosides with modified sugar moieties (ie, sugar moieties modified compared to ribose sugar moieties found in DNA and RNA). Many nucleosides with partial modification of ribose sugar have been prepared, the main purpose of which is to improve certain properties of oligonucleotides (such as affinity and/or nuclease resistance). Such modifications include the ribose ring structure (for example) that is modified by replacing it with the following structure: a hexose ring (HNA); or a bicyclic ring ( LNA); or an unlinked ribose ring that usually lacks a bond between the C2 carbon and the C3 carbon (e.g., UNA). Other sugar-modifying nucleosides include, for example, bicyclohexose nucleic acid (WO2011/017521) or tricyclic nucleic acid (WO2013/154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids. Sugar modification also includes modification by changing the substituents on the ribose ring to groups other than hydrogen or the 2'-OH groups naturally found in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4'or 5'position. Nucleosides with modified sugar moieties also include 2'modified nucleosides, such as 2'substituted nucleosides. In fact, great attention has been paid to the development of 2'substituted nucleosides, and many 2'substituted nucleosides have been found to have beneficial properties (such as enhanced nucleoside resistance and enhanced affinity) when incorporated into oligonucleotides.2' Modified nucleoside . 2'sugar modified nucleosides have substituents other than H or -OH at the 2'position (2' substituted nucleosides) or nucleosides including 2'linked diradicals, and include 2'substituted nucleosides and LNA (2'-4'double radical bridged) nucleoside. For example, 2' modified sugars can provide oligonucleotides with enhanced binding affinity and/or increased nuclease resistance. Examples of 2'substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl- RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA and 2'-F-ANA nucleosides. For other examples, see (e.g.) Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha , Chemistry and Biology 2012, 19, 937. Some 2'substitution modified nucleosides are explained below.
Figure 02_image003
Locked Nucleoside (LNA) . LNA nucleosides are modified nucleosides that include a linker group (called diradical or bridge) between C2' and C4' of the ribose ring of the nucleotide. These nucleosides are also called bridged nucleic acids or bicyclic nucleic acids (BNA) in the literature. In some embodiments, the modified nucleoside or LNA nucleoside of the oligomer of the present invention has the general structure of Formula I or II:
Figure 02_image005
or
Figure 02_image007
Formula I Formula II Wherein W is selected from -O-, -S-, -N(Ra )-, -C(Ra Rb )-, for example -O- in some embodiments; B designates a nucleobase or part of a modified nucleobase; Z is assigned to the internucleoside linkage or 5'-terminal group of adjacent nucleosides; Z* designates the internucleoside linkage or 3'-terminal group to adjacent nucleosides; X designates a group selected from the list consisting of: -C(Ra Rb )-, -C(Ra )=C(Rb )-, -C(Ra )=N-, -O-, -Si(Ra )2 -, -S-, -SO2 -, -N(Ra )-And>C=Z In some embodiments, X is selected from the group consisting of -O-, -S-, NH-, NRa Rb , -CH2 -, CRa Rb , -C(=CH2 )-And-C(=CRa Rb )- In some embodiments, X is -O-. Y designates a group selected from the group consisting of: -C(Ra Rb )-, -C(Ra )=C(Rb )-, -C(Ra )=N-, -O-, -Si(Ra )2 -, -S-, -SO2 -, -N(Ra )-And>C=Z In some embodiments, Y is selected from the group consisting of: -CH2 -, -C(Ra Rb )-, -CH2 CH2 -, -C(Ra Rb )-C(Ra Rb )-, -CH2 CH2 CH2 -, -C(Ra Rb )C(Ra Rb )C(Ra Rb )-, -C(Ra )=C(Rb )-And-C(Ra )=N- In some embodiments, Y is selected from the group consisting of: -CH2 -, -CHRa -, -CHCH3 -, CRa Rb - Or -XY- together specify a divalent linker group (also known as a free radical), and together specify a divalent linker group consisting of 1, 2, 3 or 4 groups/atoms selected from the following groups : -C(Ra Rb )-, -C(Ra )=C(Rb )-, -C(Ra )=N-, -O-, -Si(Ra )2 -, -S-, -SO2 -, -N(Ra )-And>C=Z, In some embodiments, -X-Y- designates a diradical selected from the group consisting of: -X-CH2 -, -X-CRa Rb -, -X-CHRa- , -X-C(HCH3 )- , -O-Y-, -O-CH2 -, -S-CH2 -, -NH-CH2 -, -O-CHCH3 -, -CH2 -O-CH2 , -O-CH(CH3 CH3 )-, -O-CH2 -CH2 -, OCH2 -CH2 -CH2 -, -O-CH2 OCH2 -, -O-NCH2 -, -C(=CH2 )-CH2 -, -NRa -CH2 -, N-O-CH2 , -S-CRa Rb -And-S-CHRa -. In some embodiments, -X-Y- designates -O-CH2 -Or-O-CH(CH3 )-. Wherein Z is selected from -O-, -S- and -N(Ra )-, And Ra And (when present) Rb Each independently is selected from hydrogen, optionally substituted C1-6 -Alkyl, optionally substituted C2-6 -Alkenyl, optionally substituted C2-6 -Alkynyl, hydroxyl, optionally substituted C1-6 -Alkoxy, C2-6 -Alkoxyalkyl, C2-6 -Alkenyloxy, carboxyl, C1-6 -Alkoxycarbonyl, C1-6 -Alkylcarbonyl, methanoyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl , Amine, mono-and di(C1-6 -Alkyl) amino, aminomethyl, mono- and di(C1-6 -Alkyl)-Amino-Carbonyl, Amino-C1-6 -Alkyl-aminocarbonyl, mono- and di(C1-6 -Alkyl)amino-C1-6 -Alkyl-aminocarbonyl, C1-6 -Alkyl-carbonylamino, ureido, C1-6 -Alkyloxy, sulfonyl, C1-6 -Alkylsulfonyloxy, nitro, azido, sulfanyl, C1-6 -Sulfanyl, halogen, where aryl and heteroaryl may be substituted optionally and two of the geminal substituents Ra And Rb Together, you can specify the substituted methylene group (=CH2 ), where for all opposing centers, asymmetric groups can beR orS Directional discovery. Where R1 , R2 , R3 , R5 And R5* Independently selected from the group consisting of: hydrogen, optionally substituted C1-6 -Alkyl, optionally substituted C2-6 -Alkenyl, optionally substituted C2-6 -Alkynyl, hydroxyl, C1-6 -Alkoxy, C2-6 -Alkoxyalkyl, C2-6 -Alkenyloxy, carboxyl, C1-6 -Alkoxycarbonyl, C1-6 -Alkylcarbonyl, methanoyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl , Amine, mono-and di(C1-6 -Alkyl) amino, aminomethyl, mono- and di(C1-6 -Alkyl)-Amino-Carbonyl, Amino-C1-6 -Alkyl-aminocarbonyl, mono- and di(C1-6 -Alkyl)amino-C1-6 -Alkyl-aminocarbonyl, C1-6 -Alkyl-carbonylamino, ureido, C1-6 -Alkyloxy, sulfonyl, C1-6 -Alkylsulfonyloxy, nitro, azido, sulfanyl, C1-6 -Sulfanyl, halogen, where aryl and heteroaryl may be substituted as appropriate, and two of the geminal substituents together can specify pendant oxy, pendant thio, imino or optionally substituted methylene . In some embodiments, R1 , R2 , R3 , R5 And R5* Independently selected from C1-6 Alkyl (e.g. methyl) and hydrogen. In some embodiments, R1 , R2 , R3 , R5 And R5* All are hydrogen. In some embodiments, R1 , R2 , R3 Are all hydrogen, and R5 And R5* Any of them is also hydrogen and R5 And R5* The other is not hydrogen (e.g. C1-6 Alkyl, for example methyl). In some embodiments, Ra It is hydrogen or methyl. In some embodiments, when present, Rb It is hydrogen or methyl. In some embodiments, Ra And Rb One or both of them are hydrogen. In some embodiments, Ra And Rb One of them is hydrogen and the other is not hydrogen. In some embodiments, Ra And Rb One of them is methyl and the other is hydrogen. In some embodiments, Ra And Rb All are methyl. In some embodiments, the diradical -X-Y- is -O-CH2 -, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. These LNA nucleosides are disclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160 (all of which are incorporated herein by reference), and include those commonly referred to asβ-D- Oxy LNA andα-L- Oxy LNA Nucleosides. In some embodiments, the diradical -X-Y-series -S-CH2 -, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. SuchThio LNA Nucleosides are disclosed in WO99/014226 and WO2004/046160 (which are incorporated herein by reference). In some embodiments, the diradical -X-Y- is -NH-CH2 -, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. SuchAmino LNA Nucleosides are disclosed in WO99/014226 and WO2004/046160 (which are incorporated herein by reference). In some embodiments, the diradical -X-Y- is -O-CH2 -CH2 -Or-O-CH2 -CH2 -CH2 -, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. These LNA nucleosides are disclosed in WO00/047599 and Morita et al., Bioorganic & Med. Chem. Lett. 12 73-76 (which is incorporated herein by reference), and include what is commonly referred to as 2'-O-4 'C-Ethyl bridged nucleic acid (ENA). In some embodiments, the diradical -X-Y- is -O-CH2 -, W is O, and R1 , R2 , R3 All of and R5 And R5* One of them is hydrogen, and R5 And R5* The other is not hydrogen (e.g. C1-6 Alkyl, for example methyl). Such5' replace LNA nucleosides are disclosed in WO2007/134181 (which is incorporated herein by reference). In some embodiments, the diradical-X-Y-line-O-CRa Rb -, where Ra And Rb One or both of them are not hydrogen (for example, methyl), W is O, and R1 , R2 , R3 All and one of R5 And R5* Is hydrogen, and R5 And R5* The other is not hydrogen (e.g. C1-6 Alkyl, for example methyl). SuchDouble modification LNA Nucleosides Disclosed in WO2010/077578 (which is incorporated herein by reference). In some embodiments, the diradical -X-Y- designates the divalent linker group -O-CH(CH2 OCH3 )-(2'O-Methoxyethyl bicyclic nucleic acid-Seth et al., 2010, J. Org. Chem. Vol. 75 (5), p. 1569-81). In some embodiments, the diradical -X-Y- designates the divalent linker group -O-CH(CH2 CH3 )-(2'O-Ethyl bicyclic nucleic acid-Seth et al., 2010, J. Org. Chem. Vol. 75 (5), p. 1569-81). In some embodiments, the diradical -X-Y-series -O-CHRa -, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. Such6' replace LNA nucleosides are disclosed in WO10036698 and WO07090071 (both of which are incorporated herein by reference). In some embodiments, the diradical -X-Y- is -O-CH (CH2 OCH3 )-, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. These LNA nucleosides are also known asring MOE (cMOE) and is disclosed in WO07090071. In some embodiments, the diradical -X-Y- designates the divalent linker group -O-CH(CH3 )-. -In R- or S-configuration. In some embodiments, the diradical -X-Y- together designates the divalent linker group -O-CH2 -O-CH2 -(Seth et al., 2010, J. Org. Chem). In some embodiments, the diradical -X-Y- is -O-CH (CH3 )-, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. These 6’ methyl LNA nucleosides are also known in the industrycET Nucleosides , And can be (S) cET or (R) cET stereoisomers, as disclosed in WO07090071 (β-D) and WO2010/036698 (α-L) (both are incorporated herein by reference). In some embodiments, the diradical-X-Y-line-O-CRa Rb -, where Ra Or Rb Neither is hydrogen, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. In some embodiments, Ra And Rb All are methyl. Such6' Second substitution LNA nucleosides are disclosed in WO 2009006478 (which is incorporated herein by reference). In some embodiments, the diradical-X-Y-line-S-CHRa -, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. Such6' Replace thio LNA nucleosides are disclosed in WO11156202 (which is incorporated herein by reference). In some 6' substituted thiolNA embodiments, Ra Department of methyl. In some embodiments, the diradical -X-Y- is -C(=CH2)-C(Ra Rb )- (e.g. -C(=CH2 )-CH2 -Or-C(=CH2 )-CH(CH3 )-), W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. SuchVinyl carbon LNA nucleosides are disclosed in WO08154401 and WO09067647 (both of which are incorporated herein by reference). In some embodiments, the diradical -X-Y- is -N(-ORa )- W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. In some embodiments, Ra Department C1-6 Alkyl, for example methyl. These LNA nucleosides are also referred to as N-substituted LNAs and are disclosed in WO2008/150729 (which is incorporated herein by reference). In some embodiments, the diradical -X-Y- together designates the divalent linker group -O-NRa -CH3 -(Seth et al., 2010, J. Org. Chem). In some embodiments, the diradical -X-Y-series -N(Ra )-, W is O, and R1 , R2 , R3 , R5 And R5* All are hydrogen. In some embodiments, Ra Department C1-6 Alkyl, for example methyl. In some embodiments, R5 And R5* One or both of them are hydrogen and (when substituted) R5 And R5* The other is C1-6 Alkyl (e.g. methyl). In this embodiment, R1 , R2 , R3 Both can be hydrogen, and the double radical -X-Y- can be selected from -O-CH2- or -O-C(HCRa )- (e.g. -O-C(HCH3)-). In some embodiments, the diradical system -CRa Rb -O-CRa Rb -(E.g. CH2 -O-CH2 -), W is O and R1 , R2 , R3 , R5 And R5* All are hydrogen. In some embodiments, Ra Department C1-6 Alkyl, for example methyl. These LNA nucleosides are also known as conformation restricted nucleotides (CRN) and are disclosed in WO2013036868 (which is incorporated herein by reference). In some embodiments, the diradical system -O-CRa Rb -O-CRa Rb -(E.g. O-CH2 -O-CH2 -), W is O and R1 , R2 , R3 , R5 And R5* All are hydrogen. In some embodiments, Ra Department C1-6 Alkyl, for example methyl. These LNA nucleosides are also known as COC nucleotides and are disclosed in Mitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238 (which is incorporated herein by reference). It should be recognized that unless specified, LNA nucleosides may be in the form of β-D or α-L stereoisomers. Some examples of LNA nucleosides are presented in structure Figure 1. Structure Chart 1
Figure 02_image009
As explained in the examples, in some embodiments of the present invention, the LNA nucleoside in the oligonucleotide is a β-D-oxy-LNA nucleoside.Nuclease-mediated degradation Nuclease-mediated degradation refers to an oligonucleotide capable of mediating the degradation of a complementary nucleotide sequence when it forms a duplex with this sequence. In some embodiments, oligonucleotides can function through nuclease-mediated degradation of target nucleic acids, wherein the oligonucleotides of the present invention can recruit nucleases, especially endonucleases, preferably ribonuclease ( RNase) (e.g. RNase H). Examples of oligonucleotide designs that act via a nuclease-mediated mechanism usually include a region of at least 5 or 6 DNA nucleosides and are flanked by affinity-enhancing nucleosides (e.g., gapmers, heads, etc.) on one or both sides. Polymer and tail polymer) oligonucleotides.RNase H Activity and recruitment The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when it is in the form of a duplex with a complementary RNA molecule. WO01/23613 provides in vitro methods for determining the activity of RNaseH, and these methods can be used to determine the ability to recruit RNaseH. Generally, oligonucleotides can be regarded as capable of recruiting RNase H in the following situations: when provided with complementary target nucleic acid sequences, their initial rate (as measured in pmol/l/min) is determined by the use and testing experience. Modified oligonucleotides have the same base sequence but only contain DNA monomers and have phosphorothioate linkages between all monomers in the oligonucleotides and use examples 91-95 in WO01/23613 (Incorporated herein by reference) at least 5% (eg, at least 10% or greater than 20%) of the initial rate determined by the method provided.Interstitial aggregates The term gap polymerization system as used herein refers to an antisense oligonucleotide including an oligonucleotide region (gap) that recruits RNase H, which is flanked (5' and 3') with one or more affinity-enhancing modified Nucleoside region (flank or wing). This article describes various gapmer designs and is characterized by the ability to recruit RNaseH. The head and tail polymerization systems lack a flanking oligonucleotide capable of recruiting RNase H, that is, only one end of the oligonucleotide includes an affinity-enhancing modified nucleoside. For the headmer, the 3'flanking is missing (that is, the 5'flanking includes affinity-enhancing modified nucleosides), and for the tailmer, the 5'flanking is missing (ie, the 3'flanking includes the affinity-enhancing Sex modified nucleosides).LNA Interstitial aggregates The term LNA interstitial system at least one affinity-enhancing modified nucleoside is the interstitial oligonucleotide of LNA nucleoside.Mixed wing gap polymer The term mixed wing gap polymer or mixed wing gap polymer system means that at least one flanking region includes at least one LNA nucleoside and at least one non-LNA modified nucleoside (e.g., at least one 2'substitution modified nucleoside, such as 2'-O- Alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2' -Fluorine-RNA and 2'-F-ANA nucleoside) LNA gap polymer. In some embodiments, the mixed wing gapmer has one flanking that includes only LNA nucleosides (e.g., 5'or 3') and the other flanking that includes 2'substituted modified nucleosides and optionally LNA nucleosides (respectively 3 'Or 5').Gap interrupter The term "gap interrupter oligonucleotide" is used to mean that the gap region can be maintained even if the gap region is destroyed by non-RNaseH recruitable nucleosides (interrupter nucleosides, E) (so that the gap region includes less than 5 consecutive DNA nucleosides) Interstitial aggregates recruited by RNAseH. Non-RNaseH-recruiting nucleosides are (for example) nucleosides in a 3'inward conformation, for example, the bridge between C2' and C4' in the ribose sugar ring of nucleosides is LNA in β conformation, such as β-D-oxy LNA or ScET nucleosides. The ability of gap interrupter oligonucleotides to recruit RNaseH is usually sequence or even compound specific-see Rukov et al., 2015 Nucl. Acids Res. Vol. 43, pp. 8476-8487, which reveals that recruitment provides target RNA in some cases The more specific cleaved RNaseH "gap interrupter" oligonucleotide. In some embodiments, the oligonucleotides of the present invention are gap interrupter oligonucleotides. In some embodiments, the gap interrupter oligonucleotide includes 5'-flanking (F), gap (G) 3'-flanking (F'), where the gap is recruited by non-RNaseH nucleosides (interrupter nucleoside , E) Destruction, so that the gap contains at least 3 or 4 consecutive DNA nucleosides. In some embodiments, the gap interrupter nucleoside (E) is an LNA nucleoside: wherein the bridge between C2' and C4' in the ribose sugar ring of the nucleoside is in the β conformation and is placed in the gap region, so that the gap The interrupted body LNA nucleoside flanking (5' and 3') has at least 3 (5') and 3 (3') or at least 3 (5') and 4 (3') or at least 4 (5') and 3 ( 3') DNA nucleosides, and the oligonucleotides can recruit RNaseH. The gap interrupter oligonucleotide can be represented by the following formulas: F-G-E-G-F’; especially F1-7 -G3-4 -E1 -G3-4- F’1-7 D’-F-G-F’, especially D’1-3 -F1-7 -G3-4 -E1 -G3-4 -F’1-7 F-G-F’-D’’, especially F1-7 -G3-4 -E1 -G3-4 -F’1-7 -D’’1-3 D’-F-G-F’-D’’, especially D’1-3 -F1-7 -G3-4 -E1 -G3-4 -F’1-7 -D’’1-3 The regions D’ and D’’ are as explained in the "Interstitial Polymer Design" section. In some embodiments, the gap interrupter nucleoside (E) is β-D-oxy LNA or ScET or another β-LNA nucleoside shown in the structure of Figure 1.Conjugate The term conjugate as used herein refers to an oligonucleotide covalently linked to a non-nucleotide moiety (the conjugate portion or region C or the third region), which is also referred to as an oligonucleotide conjugate. The coupling of the oligonucleotide of the invention to one or more non-nucleotide moieties can, for example, improve the pharmacology of the oligonucleotide by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments, the conjugate moiety targets the oligonucleotide to the liver. At the same time, coupling is used to reduce the activity of oligonucleotides in non-target cell types, tissues or organs, such as off-target activity or activity in non-target cell types, tissues or organs. In one embodiment of the present invention, the oligonucleotide conjugates of the present invention show improved inhibition of PD-L1 in target cells compared to unconjugated oligonucleotides. In another embodiment, compared with unconjugated oligonucleotides, the oligonucleotide conjugates of the present invention have an improved cell distribution between the liver and other organs (such as the spleen or kidney) (ie, Compared with the spleen or kidney, more conjugated oligonucleotides go to the liver). In another embodiment, the oligonucleotide conjugates of the present invention exhibit improved cellular uptake of conjugated oligonucleotides in the liver compared to unconjugated oligonucleotides. WO 93/07883 and WO2013/033230 provide suitable conjugate parts, and these patents are incorporated herein by reference. Other suitable conjugate moieties are those capable of binding to the asialoglycoprotein receptor (ASGPr). In particular, the trivalent N-acetylgalactosamine conjugate moiety is suitable for binding to ASGPr, see, for example, WO2014/076196, WO2014/207232 and WO 2014/179620 (incorporated herein by reference). The conjugate part is basically the part of the antisense oligonucleotide conjugate that is not composed of nucleic acid. Oligonucleotide conjugates and their synthesis are also reported in the following comprehensive review: Manoharan, Antisense Drug Technology, Principles, Strategies, and Applications, edited by ST Crooke, Chapter 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, the entire contents of each of them are incorporated herein by reference. In one embodiment, the non-nucleotide portion (conjugate portion) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, Toxins (such as bacterial toxins), vitamins, viral proteins (such as capsids), or combinations thereof.Connector Linking or linking system The link between one chemical group or segment of interest and another chemical group or segment of interest via one or more covalent bonds between two atoms. The conjugate moiety can be connected to the oligonucleotide directly or via a linking moiety (e.g., a linker or a conjugate). The linker is used to covalently link the third region (for example, the conjugate part, region C) to the first region (for example, oligonucleotide or adjacent nucleotide sequence, region A) that is complementary to the target nucleic acid. In some embodiments of the present invention, the conjugate or oligonucleotide conjugate of the present invention may optionally include a linker region (the second region or region B and/or region Y), which is positioned to interact with the target nucleic acid Between the complementary oligonucleotide or adjacent nucleotide sequence (region A or first region) and the conjugate part (region C or third region). Region B refers to a biologically cleavable linker that includes or consists of a physiologically unstable bond that can be normally encountered in mammals or conditions similar to those encountered in mammals Next cracking. The conditions under which the physiologically unstable linker undergoes chemical transformation (such as lysis) include chemical conditions, such as pH, temperature, oxidizing or reducing conditions or reagents, and salt concentrations found in mammalian cells or similar to those encountered in mammalian cells The salt concentration to the salt concentration. The conditions in mammalian cells also include the enzymatic activity normally present in mammalian cells (for example from proteolytic enzymes or hydrolases or nucleases). In one embodiment, the biocleavable linker is prone to S1 nuclease cleavage. In a preferred embodiment, the nuclease susceptible linker includes 1 to 10 nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, more preferably 2 to 6 nucleosides and most preferably 2 to 4 linked nucleosides). Preferably, the nucleoside is DNA or RNA. The biocleavable linker containing phosphodiester is described in more detail in WO 2014/076195 (incorporated herein by reference). Region Y refers to an oligonucleotide or contiguous nucleotide sequence (region A or the One area) connecting body. The region Y linker may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugate of the present invention can be composed of the following regional elements: A-C, A-B-C, A-B-Y-C, A-Y-B-C or A-Y-C. In some embodiments, the linker (region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group, including, for example, a C6-C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group. Treatment The term "treatment" as used herein refers to the treatment of an existing disease (for example, a disease or condition as mentioned herein) or the prevention of a disease (that is, prophylactic). It should therefore be recognized that treatments as mentioned herein may be prophylactic in some embodiments. The recovery of immune response to pathogens divides the immune response into innate immune response and adaptive immune response. The innate immune system provides an immediate but non-specific response. The adaptive immune response is activated by the innate immune response and is highly specific to specific pathogens. When pathogen-derived antigens are presented on the surface of antigen-presenting cells, immune cells of the adaptive immune response (that is, T and B lymphocytes) are activated via their antigen-specific receptors to produce pathogenic specific immune responses and produce Immune memory. Chronic viral infections (such as HBV and HCV) are associated with T cell depletion characterized by anergy of virus-specific T cells. T cell depletion has been well studied, and a review can be found, for example, in Yi et al., 2010 Immunology129, 474-481. Chronic virus infection is also related to the lowering function of NK cells, which are innate immune cells. Enhancing the viral immune response is more important for the elimination of chronic infections. The recovery of the immune response against pathogens mediated by T cells and NK cells can be evaluated by measuring proliferation, cytokine secretion and cytolytic function (Dolina et al., 2013 Molecular Therapy-Nucleic Acids, 2 e72 and herein Example 6). Detailed description of the invention The present invention relates to the use of antisense oligonucleotides and their conjugates and pharmaceutical compositions containing these substances to restore the immune response against pathogens that infect animals, especially humans. The antisense oligonucleotide conjugates of the present invention are particularly useful for combating pathogens that infect the liver, especially chronic liver infections (such as HBV). These conjugates allow targeted distribution of oligonucleotides and prevent systemic knockdown of target nucleic acids.Oligonucleotides of the invention The present invention relates to oligonucleotides capable of modulating the performance of PD-L1. This regulation can be achieved by hybridizing to a target nucleic acid encoding PD-L1 or involved in PD-L1 regulation. The target nucleic acid may be a mammalian PD-L1 sequence, for example, a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3. The target nucleic acid can be mRNA precursor, mRNA, or any RNA sequence expressed from mammalian cells that support the expression or regulation of PD-L1. The oligonucleotide of the present invention is an antisense oligonucleotide targeting PD-L1. In one aspect of the present invention, the oligonucleotide of the present invention is coupled to the conjugate portion, particularly the conjugate portion that targets the asialoglycoprotein receptor. In some embodiments, the antisense oligonucleotides of the present invention can regulate target performance by inhibiting or down-regulating the target. Preferably, the adjustment is such that the performance is inhibited by at least 20% compared with the degree of normal target performance, and more preferably by at least 30%, 40%, 50%, 60%, 70%, 80% compared with the degree of normal target performance. Or 90%. Preferably, the adjustment is such that the performance is inhibited by at least 20% compared to the degree of performance when the cell or organism is attacked by an infectious agent or treated with an agent that mimics the attack of an infectious agent (such as poly I:C or LPS) , It is better to inhibit at least 30%, 40%, 50% when compared with the performance of attacking cells or organisms by infectious agents or using reagents that simulate the attack of infectious agents (such as poly I:C or LPS) to treat cells or organisms %, 60%, 70%, 80% or 90%. In some embodiments, using KARPAS-299 or THP1 cells in vitro, the oligonucleotides of the present invention may be able to inhibit the expression of PD-L1 mRNA by at least 60% or 70%. In some embodiments, using KARPAS-299 or THP1 cells in vitro, the compound of the present invention may be able to inhibit the expression of PD-L1 protein by at least 50%. Suitably, the examples provide assays that can be used to measure PD-L1 RNA (e.g., Example 1). Target regulation is triggered by the hybridization between the adjacent nucleotide sequence of the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotides of the invention include a mismatch between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid can still be sufficient to demonstrate the desired regulation of PD-L1 performance. The reduced binding affinity derived from the mismatch can be advantageously achieved by the presence of an increased number of nucleotides in the oligonucleotide and/or the presence of an increased number of modified nucleosides capable of increasing target binding affinity ( For example, 2'modified nucleosides, including LNA) to compensate. In some embodiments, the antisense oligonucleotides of the present invention can restore pathogen-specific T cells. In some embodiments, the oligonucleotides of the invention are capable of increasing pathogen-specific T cells by at least 40%, 50%, 60%, or 70% when compared to untreated controls or controls treated with standard care. In one embodiment, the antisense oligonucleotides or conjugates of the present invention can increase HBV-specific T cells when compared with untreated controls or controls treated with standard care. Suitably, the examples provide assays that can be used to measure HBV-specific T cells (eg, T cell proliferation, cytokine secretion, and cytolytic activity). In another embodiment, the antisense oligonucleotides or conjugates of the present invention can increase HCV-specific T cells when compared with untreated controls or controls treated with standard care. In another embodiment, the antisense oligonucleotides or conjugates of the present invention can increase HDV-specific T cells when compared with untreated controls or controls treated with standard care. In some embodiments, the antisense oligonucleotides of the present invention can reduce the HBsAg content in animals or humans. In some embodiments, the oligonucleotide of the present invention can reduce the HBsAg content by at least 40%, 50%, 60%, or 70%, more preferably at least 80%, 90%, or 95% when compared with the content before treatment. %. Optimally, the oligonucleotides of the present invention can achieve seroconversion of HBsAg in HBV-infected animals or humans. One aspect of the present invention relates to an antisense oligonucleotide comprising a contiguous nucleotide sequence of 10 to 30 nucleotides in length having at least 90% complementarity with the PD-L1 target nucleic acid. In some embodiments, the oligonucleotide includes at least 90% complementary to the target nucleic acid region, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, For example, a contiguous sequence that is at least 97%, such as at least 98% or 100% complementary. In a preferred embodiment, the oligonucleotide of the present invention or its adjacent nucleotide sequence is completely complementary to the target nucleic acid region (100% complementary), or in some embodiments, it can be between the oligonucleotide and the target nucleic acid. Include one or two mismatches. In some embodiments, the oligonucleotide includes a length of 10 to 30 that is at least 90% complementary (for example, completely (or 100%) complementary) to the target nucleic acid region present in SEQ ID NO: 1 or SEQ ID NO: 2. Contiguous nucleotide sequence of two nucleotides. In some embodiments, the oligonucleotide sequence is 100% complementary to the corresponding target nucleic acid region present in SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the oligonucleotide sequence is 100% complementary to the corresponding target nucleic acid region present in SEQ ID NO: 1 and SEQ ID NO: 3. In some embodiments, the oligonucleotide or oligonucleotide conjugate includes a contiguous nucleotide sequence of 10 to 30 nucleotides in length that is at least 90% complementary (for example, 100% complementary) to the corresponding target nucleic acid region , Wherein the adjacent nucleotide sequence is complementary to a subsequence of the target nucleic acid selected from the group consisting of positions 371-3068, 5467-12107, and 15317-19511 on SEQ ID NO:1. In another embodiment, the child sequence of the target nucleic acid is selected from the group consisting of: positions 371-510, 822-1090, 1992-3068, 5467-5606, 6470-12107, 15317-15720 on SEQ ID NO: 1. , 15317-18083, 18881-19494 and 1881-19494. In a preferred embodiment, the child sequence of the target nucleic acid is selected from the group consisting of: positions 7300-7333, 8028-8072, 9812-9859, 11787-11873, and 15690-15735 on SEQ ID NO:1. In some embodiments, the oligonucleotide or oligonucleotide conjugate includes a length of 10 to 30 that is at least 90% complementary (for example, 100% complementary) to the corresponding target nucleic acid region present in SEQ ID NO: 1. The contiguous nucleotide sequence of nucleotides, wherein the target nucleic acid region is selected from the group consisting of regions a1 to a449 in Table 4. Table 4: Regions of SEQ ID NO 1 that can be targeted by the oligonucleotides of the present invention Reg. a Position in SEQ ID NO 1 length Reg.a Position in SEQ ID NO 1 length Reg.a Position in SEQ ID NO 1 length starting point end starting point end starting point end a1 51 82 32 a151 6994 7020 27 a301 13092 13115 twenty four a2 87 116 30 a152 7033 7048 16 a302 13117 13134 18 a3 118 133 16 a153 7050 7066 17 a303 13136 13169 34 a4 173 206 34 a154 7078 7094 17 a304 13229 13249 twenty one a5 221 287 67 a155 7106 7122 17 a305 13295 13328 34 a6 304 350 47 a156 7123 7144 twenty two a306 13330 13372 43 a7 354 387 34 a157 7146 7166 twenty one a307 13388 13406 19 a8 389 423 35 a158 7173 7193 twenty one a308 13408 13426 19 a9 425 440 16 a159 7233 7291 59 a309 13437 13453 17 a10 452 468 17 a160 7300 7333 34 a310 13455 13471 17 a11 470 484 15 a161 7336 7351 16 a311 13518 13547 30 a12 486 500 15 a162 7353 7373 twenty one a312 13565 13,597 33 a13 503 529 27 a163 7375 7412 38 a313 13603 13620 18 a14 540 574 35 a164 7414 7429 16 a314 13630 13663 34 a15 576 649 74 a165 7431 7451 twenty one a315 13665 13679 15 a16 652 698 47 a166 7453 7472 20 a316 13706 13725 20 a17 700 750 51 a167 7474 7497 twenty four a317 13727 13,774 48 a18 744 758 15 a168 7517 7532 16 a318 13784 13821 38 a19 774 801 28 a169 7547 7601 55 a319 13,831 13878 48 a20 805 820 16 a170 7603 7617 15 a320 13881 13940 60 a21 827 891 65 a171 7632 7647 16 a321 13959 14013 55 a22 915 943 29 a172 7649 7666 18 a322 14015 14031 17 a23 950 982 33 a173 7668 7729 62 a323 14034 14049 16 a24 984 1000 17 a174 7731 7764 34 a324 14064 14114 51 a25 1002 1054 53 a175 7767 7817 51 a325 14116 14226 111 a26 1060 1118 59 a176 7838 7860 twenty three a326 14229 14276 48 a27 1124 1205 82 a177 7862 7876 15 a327 14292 14306 15 a28 1207 1255 49 a178 7880 7944 65 a328 14313 14384 72 a29 1334 1349 16 a179 7964 8012 49 a329 14386 14408 twenty three a30 1399 1425 27 a180 8028 8072 45 a330 14462 14481 20 a31 1437 1458 twenty two a181 8086 8100 15 a331 14494 14519 26 a32 1460 1504 45 a182 8102 8123 twenty two a332 14557 14577 twenty one a33 1548 1567 20 a183 8125 8149 25 a333 14608 14628 twenty one a34 1569 1586 18 a184 8151 8199 49 a334 14,646 14668 twenty three a35 1608 1662 55 a185 8218 8235 18 a335 14,680 14,767 88 a36 1677 1700 twenty four a186 8237 8276 40 a336 14,765 14,779 15 a37 1702 1721 20 a187 8299 8344 46 a337 14815 14,844 30 a38 1723 1745 twenty three a188 8346 8436 91 a338 14,848 14925 78 a39 1768 1794 27 a189 8438 8470 33 a339 14,934 14,976 43 a40 1820 1835 16 a190 8472 8499 28 a340 14,978 15009 32 a41 1842 1874 33 a191 8505 8529 25 a341 15013 15057 45 a42 1889 1979 91 a192 8538 8559 twenty two a342 15064 15091 28 a43 1991 2011 twenty one a193 8562 8579 18 a343 15094 15140 47 a44 2013 2038 26 a194 8581 8685 105 a344 15149 15165 17 a45 2044 2073 30 a195 8688 8729 42 a345 15162 15182 twenty one a46 2075 2155 81 a196 8730 8751 twenty two a346 15184 15198 15 a47 2205 2228 twenty four a197 8777 8800 twenty four a347 15200 15221 twenty two a48 2253 2273 twenty one a198 8825 8865 41 a348 15232 15247 16 a49 2275 2303 29 a199 8862 8894 33 a349 15250 15271 twenty two a50 2302 2333 32 a200 8896 8911 16 a350 15290 15334 45 a51 2335 2366 32 a201 8938 8982 45 a351 15336 15369 34 a52 2368 2392 25 a202 8996 9045 50 a352 15394 15416 twenty three a53 2394 2431 38 a203 9048 9070 twenty three a353 15433 15451 19 a54 2441 2455 15 a204 9072 9139 68 a354 15453 15491 39 a55 2457 2494 38 a205 9150 9168 19 a355 15496 15511 16 a56 2531 2579 49 a206 9170 9186 17 a356 15520 15553 34 a57 2711 2732 twenty two a207 9188 9202 15 a357 15555 15626 72 a58 2734 2757 twenty four a208 9204 9236 33 a358 15634 15652 19 a59 2772 2786 15 a209 9252 9283 32 a359 15655 15688 34 a60 2788 2819 32 a210 9300 9331 32 a360 15690 15735 46 a61 2835 2851 17 a211 9339 9354 16 a361 15,734 15,764 31 a62 2851 2879 29 a212 9370 9398 29 a362 15,766 15,787 twenty two a63 2896 2912 17 a213 9400 9488 89 a363 15803 15,819 17 a64 2915 2940 26 a214 9490 9537 48 a364 15846 15899 54 a65 2944 2973 30 a215 9611 9695 85 a365 15901 15934 34 a66 2973 2992 20 a216 9706 9721 16 a366 15936 15962 27 a67 2998 3016 19 a217 9723 9746 twenty four a367 15964 15985 twenty two a68 3018 3033 16 a218 9748 9765 18 a368 15,987 16023 37 a69 3036 3051 16 a219 9767 9788 twenty two a369 16025 16061 37 a70 3114 3139 26 a220 9794 9808 15 a370 16102 16122 twenty one a71 3152 3173 twenty two a221 9812 9859 48 a371 16134 16183 50 a72 3181 3203 twenty three a222 9880 9913 34 a372 16185 16281 97 a73 3250 3271 twenty two a223 9923 9955 33 a373 16283 16,298 16 a74 3305 3335 31 a224 9966 10007 42 a374 16305 16323 19 a75 3346 3363 18 a225 10009 10051 43 a375 16,325 16356 32 a76 3391 3446 56 a226 10053 10088 36 a376 16362 16404 43 a77 3448 3470 twenty three a227 10098 10119 twenty two a377 16406 16456 51 a78 3479 3497 19 a228 10133 10163 31 a378 16494 16523 30 a79 3538 3554 17 a229 10214 10240 27 a379 16536 16562 27 a80 3576 3597 twenty two a230 10257 10272 16 a380 16564 16580 17 a81 3603 3639 37 a231 10281 10298 18 a381 16582 16637 56 a82 3663 3679 17 a232 10300 10318 19 a382 16631 16649 19 a83 3727 3812 86 a233 10339 10363 25 a383 16,655 16701 47 a84 3843 3869 27 a234 10409 10426 18 a384 16737 16781 45 a85 3874 3904 31 a235 10447 10497 51 a385 16783 16804 twenty two a86 3926 3955 30 a236 10499 10529 31 a386 16832 16907 76 a87 3974 3993 20 a237 10531 10546 16 a387 16934 16965 32 a88 3995 4042 48 a238 10560 10580 twenty one a388 16,972 17035 64 a89 4053 4073 twenty one a239 10582 10596 15 a389 17039 17069 31 a90 4075 4123 49 a240 10600 10621 twenty two a390 17072 17109 38 a91 4133 4157 25 a241 10623 10664 42 a391 17135 17,150 16 a92 4158 4188 31 a242 10666 10685 20 a392 17,167 17,209 43 a93 4218 4250 33 a243 10717 10773 57 a393 17211 17,242 32 a94 4277 4336 60 a244 10775 10792 18 a394 17244 17,299 56 a95 4353 4375 twenty three a245 10794 10858 65 a395 17,304 17344 41 a96 4383 4398 16 a246 10874 10888 15 a396 17346 17,400 55 a97 4405 4446 42 a247 10893 10972 80 a397 17,447 17,466 20 a98 4448 4464 17 a248 10974 10994 twenty one a398 17,474 17539 66 a99 4466 4493 28 a249 10996 11012 17 a399 17,561 17,604 44 a100 4495 4558 64 a250 11075 11097 twenty three a400 17610 17,663 54 a101 4571 4613 43 a251 11099 11124 26 a401 17,681 17763 83 a102 4624 4683 60 a252 11140 11157 18 a402 17793 17,810 18 a103 4743 4759 17 a253 11159 11192 34 a403 17812 17852 41 a104 4761 4785 25 a254 11195 11226 32 a404 17,854 17,928 75 a105 4811 4858 48 a255 11235 11261 27 a405 17,941 18005 65 a106 4873 4932 60 a256 11279 11337 59 a406 18007 18035 29 a107 4934 4948 15 a257 11344 11381 38 a407 18041 18077 37 a108 4955 4974 20 a258 11387 11411 25 a408 18085 18146 62 a109 4979 5010 32 a259 11427 11494 68 a409 18163 18177 15 a110 5012 5052 41 a260 11496 11510 15 a410 18179 18207 29 a111 5055 5115 61 a261 11512 11526 15 a411 18209 18228 20 a112 5138 5166 29 a262 11528 11551 twenty four a412 18230 18266 37 a113 5168 5198 31 a263 11570 11592 twenty three a413 18268 18285 18 a114 5200 5222 twenty three a264 11594 11634 41 a414 18287 18351 65 a115 5224 5284 61 a265 11664 11684 twenty one a415 18365 18395 31 a116 5286 5302 17 a266 11699 11719 twenty one a416 18402 18432 31 a117 5317 5332 16 a267 11721 11746 26 a417 18434 18456 twenty three a118 5349 5436 88 a268 11753 11771 19 a418 18502 18530 29 a119 5460 5512 53 a269 11787 11873 87 a419 18545 18590 46 a120 5514 5534 twenty one a270 11873 11905 33 a420 18603 18621 19 a121 5548 5563 16 a271 11927 11942 16 a421 18623 18645 twenty three a122 5565 5579 15 a272 11946 11973 28 a422 18651 18708 58 a123 5581 5597 17 a273 11975 11993 19 a423 18710 18729 20 a124 5600 5639 40 a274 12019 12114 96 a424 18731 18758 28 a125 5644 5661 18 a275 12116 12135 20 a425 18760 18788 29 a126 5663 5735 73 a276 12137 12158 twenty two a426 18799 18859 61 a127 5737 5770 34 a277 12165 12192 28 a427 18861 18926 66 a128 5778 5801 twenty four a278 12194 12216 twenty three a428 18928 18980 53 a129 5852 5958 107 a279 12218 12246 29 a429 19001 19018 18 a130 6007 6041 35 a280 12262 12277 16 a430 19034 19054 twenty one a131 6049 6063 15 a281 12283 12319 37 a431 19070 19092 twenty three a132 6065 6084 20 a282 12334 12368 35 a432 19111 19154 44 a133 6086 6101 16 a283 12370 12395 26 a433 19191 19213 twenty three a134 6119 6186 68 a284 12397 12434 38 a434 19215 19240 26 a135 6189 6234 46 a285 12436 12509 74 a435 19255 19356 102 a136 6236 6278 43 a286 12511 12543 33 a436 19358 19446 89 a137 6291 6312 twenty two a287 12545 12565 twenty one a437 19450 19468 19 a138 6314 6373 60 a288 12567 12675 109 a438 19470 19512 43 a139 6404 6447 44 a289 12677 12706 30 a439 19514 19541 28 a140 6449 6482 34 a290 12708 12724 17 a440 19543 19568 26 a141 6533 6555 twenty three a291 12753 12768 16 a441 19570 19586 17 a142 6562 6622 61 a292 12785 12809 25 a442 19588 19619 32 a143 6624 6674 51 a293 12830 12859 30 a443 19683 19739 57 a144 6679 6762 84 a294 12864 12885 twenty two a444 19741 19777 37 a145 6764 6780 17 a295 12886 12916 31 a445 19779 19820 42 a146 6782 6822 41 a296 12922 12946 25 a446 19822 19836 15 a147 6824 6856 33 a297 12948 12970 twenty three a447 19838 19911 74 a148 6858 6898 41 a298 12983 13003 twenty one a448 19913 19966 54 a149 6906 6954 49 a299 13018 13051 34 a449 19968 20026 59 a150 6969 6992 twenty four a300 13070 13090 twenty one In some embodiments, the oligonucleotide or contiguous nucleotide sequence is complementary to the target nucleic acid region, wherein the target nucleic acid region is selected from the group consisting of a7, a26, a43, a119, a142, a159, a160, a163, a169, a178, a179, a180, a189, a201, a202, a204, a214, a221, a224, a226, a243, a254, a258, 269, a274, a350, a360, a364, a365, a370, a372, a381, a383, a386, a389, a400, a427, a435 and a438. In a preferred embodiment, the oligonucleotide or adjacent nucleotide sequence is complementary to the target nucleic acid region, wherein the target nucleic acid region is selected from the group consisting of a160, a180, a221, a269 and a360. In some embodiments, the oligonucleotides of the present invention include or consist of 8 to 35 nucleotides (length), for example, 9 to 30, such as 10 to 22, such as 11 to 20, such as 12 to 18, such as 13 to 17 or 14 to 16 contiguous nucleotides. In a preferred embodiment, the oligonucleotide includes or consists of 16 to 20 nucleotides (length). It should be understood that any range given herein includes the end points of the range. Therefore, if it is mentioned that an oligonucleotide contains 10 to 30 nucleotides, then 10 and 30 nucleotides are included. In some embodiments, the contiguous nucleotide sequence includes 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides (length) or consist of them. In a preferred embodiment, the oligonucleotide includes or consists of 16, 17, 18, 19, or 20 nucleotides (length). In some embodiments, the oligonucleotide or contiguous nucleotide sequence includes or consists of a sequence selected from the group consisting of the sequences listed in Table 5. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 and selected from the group consisting of SEQ ID NO: 5 to 743 (see the motif sequence listed in Table 5). The sequence has or consists of nucleotides (length) with at least 90% identity, preferably 100% identity. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 sequences that have at least 90% identity with a sequence selected from the group consisting of SEQ ID NO: 5 to 743 and 771, preferably 100% identical nucleotides (length) or consist of them. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 nucleotides with at least 90% identity, preferably 100% identity, with a sequence selected from the group consisting of (Length) or consisting of: SEQ ID NO: 6, 8, 9, 13, 41, 42, 58, 77, 92, 111, 128, 151, 164, 166, 169, 171, 222, 233, 245, 246, 250, 251, 252, 256, 272, 273, 287, 292, 303, 314, 318, 320, 324, 336, 342, 343, 344, 345, 346, 349, 359, 360, 374, 408, 409, 415, 417, 424, 429, 430, 458, 464, 466, 474, 490, 493, 512, 519, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 640, 645, 650, 651, 652, 653, 658, 659, 660, 665, 678, 679, 680, 682, 683, 684, 687, 694, 706, 716, 728, 733, 734 and 735. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 nucleotides (length) with at least 90% identity, preferably 100% identity, with SEQ ID NO: 287 Or consist of it. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 nucleotides (length) with at least 90% identity, preferably 100% identity with SEQ ID NO: 342 Or consist of it. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 nucleotides (length) with at least 90% identity, preferably 100% identity with SEQ ID NO: 640 Or consist of it. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 nucleotides (length) with at least 90% identity, preferably 100% identity with SEQ ID NO: 466 Or consist of it. In some embodiments, the antisense oligonucleotide or contiguous nucleotide sequence includes 10 to 30 nucleotides (length) with at least 90% identity, preferably 100% identity with SEQ ID NO: 566 Or consist of it. In the embodiment where the oligonucleotide is longer than the contiguous nucleotide sequence (which is complementary to the target nucleic acid), the motif sequence in Table 5 forms part of the contiguous nucleotide sequence of the antisense oligonucleotide of the present invention. In some embodiments, the sequence of the oligonucleotide is equivalent to the adjacent nucleotide sequence (for example, if no biocleavable linker is added). It should be understood that the contiguous nucleobase sequence (motif sequence) can be modified to, for example, increase nuclease resistance and/or binding affinity for the target nucleic acid. Modifications are described in the definitions and "Oligonucleotide Design" section. Table 5 lists the preferred designs for each motif sequence. Oligonucleotide design Oligonucleotide design refers to the nucleoside sugar modification pattern in the oligonucleotide sequence. The oligonucleotides of the present invention include sugar-modified nucleosides and may also include DNA or RNA nucleosides. In some embodiments, oligonucleotides include sugar modified nucleosides and DNA nucleosides. The incorporation of modified nucleosides into the oligonucleotides of the present invention can enhance the affinity of the oligonucleotides for the target nucleic acid. In this case, the modified nucleoside can be referred to as an affinity-enhancing modified nucleotide, and the modified nucleoside can also be referred to as a unit. In one embodiment, the oligonucleotide includes at least 1 modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, At least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In one embodiment, the oligonucleotide includes 1 to 10 modified nucleosides, such as 2 to 8 modified nucleosides, such as 3 to 7 modified nucleosides, such as 4 to 6 modified nucleosides, such as 3, 4, 5, 6, or 7 modified nucleosides. In one embodiment, the oligonucleotide includes one or more sugar-modified nucleosides, such as 2&apos; sugar-modified nucleosides. Preferably, the oligonucleotide of the present invention includes one or more 2'sugar-modified nucleosides independently selected from the group consisting of: 2'-O-alkyl-RNA, 2'-O-methyl- RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro -ANA and LNA nucleosides. Even more preferably, one or more modified nucleosides are locked nucleic acids (LNA). In another embodiment, the oligonucleotide includes at least one modified internucleoside linkage. In a preferred embodiment, all internucleoside linkages in adjacent nucleotide sequences are phosphorothioate or borane phosphate internucleoside linkages. In some embodiments, all internucleotide linkages in the contiguous sequence of the oligonucleotide are phosphorothioate linkages. In some embodiments, the oligonucleotides of the invention include at least one LNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7 or 8 LNA nucleosides, such as 2 to 6 LNA nucleosides, such as 3 to 7 LNA nucleosides, 4 to 6 LNA nucleosides or 3, 4, 5, 6 or 7 LNA nucleosides. In some embodiments, at least 75% of the modified nucleosides in the oligonucleotide are LNA nucleosides, for example, 80%, such as 85%, such as 90% of the modified nucleosides are LNA nucleosides. In another embodiment, all modified nucleosides in the oligonucleotide are LNA nucleosides. In another embodiment, the oligonucleotide may include β-D-oxy-LNA and one or more of the following LNA nucleosides: thio-LNA, amino-LNA, oxy-LNA and/or ENA (in β-D or α-L configuration or a combination thereof). In another embodiment, all LNA cytosine units are 5-methyl-cytosine. In a preferred embodiment, the oligonucleotide or adjacent nucleotide sequence has at least 1 LNA nucleoside at the 5'end of the nucleotide sequence and at least 2 LNA nucleosides at the 3'end. In some embodiments, the oligonucleotides of the present invention include at least one modified nucleoside that is a 2'-MOE-RNA nucleoside, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-MOE-RNA nucleosides. '-MOE-RNA nucleoside. In some embodiments, at least one of the modified nucleosides is 2'-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2'-fluoro-DNA nuclei Glycosides. In some embodiments, the oligonucleotides of the invention include at least one LNA nucleoside and at least one 2&apos; substitution modified nucleoside. In some embodiments of the present invention, oligonucleotides include 2'sugar modified nucleosides and DNA units. Preferably, the oligonucleotide includes LNA and DNA nucleosides (units). Preferably, the total number of combinations of LNA and DNA units is 8-30 (such as 10-25, preferably 12-22, such as 12-18, even more preferably 11-16). In some embodiments of the present invention, the nucleotide sequence of the oligonucleotide (for example, the contiguous nucleotide sequence) is composed of at least one or two LNA nucleosides and the remaining nucleosides are DNA units. In some embodiments, oligonucleotides include only LNA nucleosides and natural nucleosides (e.g. RNA or DNA, optimally DNA nucleosides), and optionally include modified internucleoside linkages (e.g. phosphorothioate) . In one embodiment of the present invention, the oligonucleotide of the present invention can recruit RNase H. The structural design of the oligonucleotide of the present invention can be selected from gapmers, gap interrupters, headmers and tailmers. Interstitial polymer design In a preferred embodiment, the oligonucleotide of the present invention has an interstitial polymer design or structure, and the interstitial polymer design or structure is also only referred to as "interstitial polymer" herein. In the gapmer structure, the oligonucleotide includes at least three different structural regions: 5'-flanking, gap, and 3'-flanking (F-G-F', with "5 -> 3" orientation). In this design, when the oligonucleotide and the target nucleic acid are in the form of a duplex, the flanking regions F and F'(also called wing regions) include adjacent sequence segments of modified nucleosides complementary to the PD-L1 target nucleic acid , And the gap region G includes a contiguous sequence of nucleotides capable of recruiting nucleases, preferably endonucleases (such as RNase, such as RNase H). Nucleosides capable of recruiting nucleases, especially RNase H, can be selected from the group consisting of DNA, α-L-oxy-LNA, 2'-fluoro-ANA and UNA. The regions F and F'at the 5'and 3'ends of the flanking region G preferably include non-nuclease-recruiting nucleosides (nucleosides with a 3'inward structure), more preferably one or more affinity-enhancing nucleosides Modified nucleosides. In some embodiments, the 3&apos; flanking includes at least one LNA nucleoside, preferably at least 2 LNA nucleosides. In some embodiments, the 5&apos; flank includes at least one LNA nucleoside. In some embodiments, the 5'and 3'flanking regions include LNA nucleosides. In some embodiments, all nucleosides in the flanking region are LNA nucleosides. In other embodiments, the flanking region may include LNA nucleosides and other nucleosides (mixed flanking), such as DNA nucleosides and/or non-LNA modified nucleosides (e.g., 2&apos; substituted nucleosides). In this case, the gap is defined as at least 5 RNase H recruiting properties flanked by affinity-enhancing modified nucleosides, preferably LNAs (such as β-D-oxy-LNA) at the 5'and 3'ends. The adjacent sequence of nucleosides (nucleosides with 2'inward structure, preferably DNA). Therefore, the nucleosides of the 5'flanking region and the 3'flanking region adjacent to the gap region are modified nucleosides, preferably non-nuclease-recruiting nucleosides. Region F The region F (5' flanking or 5'wing) connected to the '5 end of the region G includes or contains at least one modified nucleoside (e.g. at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 Modified nucleosides) or consist of them. In one embodiment, the region F includes 1 to 7 modified nucleosides (e.g., 2 to 6 modified nucleosides, such as 2 to 5 modified nucleosides, such as 2 to 4 modified nucleosides, such as 1 To 3 modified nucleosides, for example 1, 2, 3 or 4 modified nucleosides) or consist of them. The F region is defined as having at least one modified nucleoside at the 5'end and 3'end of the region. In some embodiments, the modified nucleosides in region F have a 3&apos; inward structure. In one embodiment, one or more of the modified nucleosides in region F are 2&apos; modified nucleosides. In one embodiment, all nucleosides in region F are 2'modified nucleosides. In another embodiment, in addition to 2'modified nucleosides, region F also includes DNA and/or RNA. The flanking including DNA and/or RNA is characterized by having 2'modified nucleosides at the 5'end and 3'end (adjacent to the G region) of the F region. In one embodiment, the region F includes DNA nucleosides, such as 1 to 3 adjacent DNA nucleosides, such as 1 to 3 or 1 to 2 adjacent DNA nucleosides. The DNA nucleosides in the flanks should preferably not be able to recruit RNase H. In some embodiments, the 2'modified nucleosides and DNA and/or RNA nucleosides in the F region alternate with 1 to 3 2'modified nucleosides and 1 to 3 DNA and/or RNA nucleosides. These flanks can also be referred to as alternate flanks. The length of the 5'flanking (region F) in an oligonucleotide with alternate flanking can be 4 to 10 nucleosides, such as 4 to 8, such as 4 to 6 nucleosides, such as 4, 5, 6 or 7 A modified nucleoside. In some embodiments, only the 5&apos; flanking of the oligonucleotide is alternating. Specific examples of region F with alternating nucleosides are as follows: 2'1-3 -N’1-4 -2'1-3 2'1-2 -N’1-2 -2'1-2 -N’1-2 -2'1-2 Wherein 2'indicates a modified nucleoside and N'is RNA or DNA. In some embodiments, all modified nucleosides in the alternate flanks are LNA and N'are DNA. In another embodiment, one or more 2'modified nucleosides in region F are selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino- DNA unit, 2'-fluoro-DNA unit, 2'-alkoxy-RNA, MOE unit, LNA unit, arabinose nucleic acid (ANA) unit and 2'-fluoro-ANA unit. In some embodiments, the F region includes LNA and 2&apos; substituted modified nucleosides. These forms are commonly referred to as mixed wing or mixed flanking oligonucleotides. In an embodiment of the present invention, all modified nucleosides in region F are LNA nucleosides. In another embodiment, all nucleosides in region F are LNA nucleosides. In another embodiment, the LNA nucleosides in region F are independently selected from the group consisting of: oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA (in β-D or α -L configuration or a combination). In a preferred embodiment, the region F includes at least 1β-D-oxy LNA unit at the 5'end of the adjacent sequence. Region G Region G (gap region) preferably includes or contains at least 4 (such as at least 5, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, At least 15 or at least 16) capable of recruiting or consisting of consecutive nucleosides of the aforementioned nucleases, especially RNaseH. In another embodiment, the region G includes, contains, or consists of, 5 to 12, or 6 to 10, or 7 to 9, for example, 8 consecutive nucleotide units capable of recruiting the aforementioned nucleases. The nucleoside unit capable of recruiting nuclease in region G is selected from the group consisting of DNA, α-L-LNA, C4' alkylated DNA in one embodiment (such as PCT/EP2009/050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300 (both are incorporated herein by reference)), arabinose-derived nucleosides (such as ANA and 2'F-ANA) (Mangos etc.) People, 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst., 2009, 10, 1039, this document is incorporated by reference Into this article). UNA is a non-locked nucleic acid, in which the bond between C2 and C3 of ribose is usually removed to form non-locked "sugar" residues. In another embodiment, at least one nucleoside unit in region G is a DNA nucleoside unit, such as 1 to 18 DNA units, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16 or 17 DNA units, preferably 2 to 17 DNA units, such as 3 to 16 DNA units, such as 4 to 15 DNA units, such as 5 to 14 DNA units, For example, 6 to 13 DNA units, such as 7 to 12 DNA units, such as 8 to 11 DNA units, more preferably 8 to 17 DNA units or 9 to 16 DNA units, 10 to 15 DNA units or 11 to 13 DNA units, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 DNA units. In some embodiments, region G is composed of 100% DNA units. In other embodiments, region G can be composed of a mixture of DNA and other nucleosides that can mediate RNase H cleavage. Region G may be composed of at least 50% DNA, more preferably 60%, 70% or 80% DNA, and even more preferably 90% or 95% DNA. In another embodiment, at least one nucleoside unit in region G is an α-L-LNA nucleoside unit, such as at least one α-L-LNA, such as 2, 3, 4, 5, 6, 7, 8 or 9 α-L-LNA. In another embodiment, the region G includes at least one α-L-LNA which is α-L-oxy-LNA. In another embodiment, region G includes a combination of DNA and α-L-LNA nucleoside units. In some embodiments, the nucleosides in region G have a 2&apos; inward structure. In some embodiments, region G may include gap interrupter nucleosides, thereby generating gap interrupter oligonucleotides, which are capable of recruiting RNase H. The region F'(3' flanking or 3'wing) connected to the '3 end of the region G includes or contains at least one modified nucleoside (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, At least 7 modified nucleosides) or consist of them. In one embodiment, the region F'includes 1 to 7 modified nucleosides (e.g., 2 to 6 modified nucleosides, e.g., 2 to 4 modified nucleosides, e.g., 1 to 3 modified nucleosides, such as 1, 2, 3 or 4 modified nucleosides) or consist of them. The F'region is defined as having at least one modified nucleoside at the 5'end and the 3'end of the region. In some embodiments, the modified nucleosides in region F'have a 3'internal structure. In one embodiment, one or more modified nucleosides in the region F'are 2'modified nucleosides. In one embodiment, all nucleosides in region F'are 2'modified nucleosides. In one embodiment, one or more modified nucleosides in the region F'are 2'modified nucleosides. In one embodiment, all nucleosides in region F'are 2'modified nucleosides. In another embodiment, in addition to the 2'modified nucleosides, the region F'also includes DNA or RNA. The flanking including DNA or RNA is characterized by having 2'modified nucleosides at the 5'end (adjacent to the G region) and the 3'end of the F'region. In one embodiment, the region F'includes DNA nucleosides, such as 1 to 4 adjacent DNA nucleosides, such as 1 to 3 or 1 to 2 adjacent DNA nucleosides. The DNA nucleosides in the flanks should preferably not be able to recruit RNase H. In some embodiments, the 2'modified nucleosides and DNA and/or RNA nucleosides in the F'region alternate with 1 to 3 2'modified nucleosides and 1 to 3 DNA and/or RNA nucleosides. The flanks can also be referred to as alternate flanks. The length of the 3'flanking (region F') in an oligonucleotide with alternate flanking can be 4 to 10 nucleosides, for example 4 to 8, for example 4 to 6 nucleosides, for example 4, 5, 6 or 7 modified nucleosides. In some embodiments, only the 3&apos; flanking of the oligonucleotide is alternating. Specific examples of the region F'with alternate nucleosides are as follows: 2'1-2 -N’1-4 -2'1-4 2'1-2 -N’1-2 -2'1-2 -N’1-2 -2'1-2 Wherein 2'indicates a modified nucleoside and N'is RNA or DNA. In some embodiments, all modified nucleosides in the alternate flanks are LNA and N'are DNA. In another embodiment, the modified nucleoside in region F'is selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2 '-Fluoro-DNA unit, 2'-alkoxy-RNA, MOE unit, LNA unit, arabino nucleic acid (ANA) unit and 2'-fluoro-ANA unit. In some embodiments, the F&apos; region includes LNA and 2&apos; substituted modified nucleosides. These forms are commonly referred to as mixed wing or mixed flanking oligonucleotides. In an embodiment of the present invention, all modified nucleosides in region F'are LNA nucleosides. In another embodiment, all nucleosides in region F'are LNA nucleosides. In another embodiment, the LNA nucleosides in region F'are independently selected from the group consisting of: oxy-LNA, thio-LNA, amino-LNA, cET and/or ENA (in β-D or α-L configuration or a combination thereof). In a preferred embodiment, the region F'has at least 2β-D-oxy LNA units at the 3'end of the adjacent sequence. Area D’ and D’’ Area D’ and D’’ can be connected to the 5’ end of the area F or the 3’ end of the area F’, respectively. The area D'or D'' is optional. The region D'or D'' can independently include 0 to 5 (for example, 1 to 5, for example, 2 to 4, for example, 0, 1, 2, 3, 4, or 5) and can be complementary or non-complementary to the target nucleic acid The other nucleotides. In this regard, in some embodiments, the oligonucleotides of the present invention may include adjacent nucleotide sequences flanking other nucleotides at the 5'and/or 3'ends that can regulate the target. These other nucleotides can be used as nuclease-susceptible biocleavable linkers (see the definition of linkers). In some embodiments, other 5'and/or 3'nucleosides are linked to phosphodiester links, and they can be DNA or RNA. In another embodiment, other 5&apos; and/or 3&apos; end nucleosides may, for example, be included to enhance nuclease stability or to facilitate synthesis of modified nucleosides. In one embodiment, the oligonucleotide of the present invention includes a region D'and/or D'at the 5'or 3'end of the adjacent nucleotide sequence. In another embodiment, the D'and/or D'region is composed of 1 to 5 DNA or RNA nucleosides linked to a phosphodiester that is not complementary to the target nucleic acid. The gapmer oligonucleotide of the present invention can be represented by the following formulas: 5’-F-G-F’-3’; especially F1-7 -G4-12 -F’1-7 5’-D’-F-G-F’-3’, especially D’1-3 -F1-7 -G4-12 -F’1-7 5’-F-G-F’-D’’-3’, especially F1-7 -G4-12 -F’1-7 -D’’1-3 5’-D’-F-G-F’-D’-3’’, especially D’1-3 -F1-7 -G4-12 -F’1-7 -D’’1-3 The preferred amounts and types of nucleosides in regions F, G and F', D'and D'' have been described above. The oligonucleotide conjugate of the present invention has a region C covalently linked to the 5'or 3'end of the oligonucleotide, especially the gapmer oligonucleotide presented above. In one embodiment, the oligonucleotide conjugate of the present invention includes an oligonucleotide having the formula 5'-D'-FG-F'-3' or 5'-FG-F'-D''-3' Nucleosides, where regions F and F'independently include 1-7 modified nucleosides, G is 6-16 regions capable of recruiting RNaseH nucleosides, and region D'or D'' includes 1-5 phosphate diphosphates Ester-linked nucleosides. Preferably, the region D'or D'' is present at the end of the conjugated oligonucleotide covering the conjugate portion. Examples of oligonucleotides with alternating flanks can be represented by the following formulas: 2'1-3 -N’1-4 -2'1-3 -G6-12 -2'1-2 -N’1-4 -2'1-4 2'1-2 -N’1-2 -2'1-2 -N’1-2 -2'1-2 -G6-12 -2'1-2 -N’1-2 -2'1-2 -N’1-2 -2'1-2 F-G6-12 -2'1-2 -N’1-4 -2'1-4 F-G6-12 -2'1-2 -N’1-2 -2'1-2 -N’1-2 -2'1-2 2'1-3 -N’1-4 -2'1-3 -G6-12 -F’ 2'1-2 -N’1-2 -2'1-2 -N1-2 -2'1-2 -G6-12 -F’ Wherein the flanks are indicated by F or F', which only contain 2'modified nucleosides (e.g. LNA nucleosides). The preferred numbers and types of nucleosides in alternate regions and regions F, G and F', D'and D'' have been described above. In some embodiments, the oligonucleotide is a gapmer composed of 16, 17, 18, 19, 20, 21, 22 nucleotides (length), which is in the form of a double helix with the PD-L1 target nucleic acid. When, each of regions F and F'is independently composed of 1, 2, 3, or 4 modified nucleoside units complementary to the PD-L1 target nucleic acid, and region G is composed of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 nucleoside units capable of recruiting nucleases are composed of nucleoside units, and the region D'is composed of 2 phosphodiester linked DNA. In another embodiment, the oligonucleotide is a gapmer, where each of regions F and F'independently consists of 3, 4, 5, or 6 modified nucleoside units (e.g., containing 2'-O -Methoxyethyl-ribose (2'-MOE) nucleoside unit or 2'-fluoro-deoxyribose-containing nucleoside unit and/or LNA unit), and region G is composed of 8, 9, 10 , 11, 12, 13, 14, 15, 16 or 17 nucleoside units (e.g. DNA unit or other nuclease-recruiting nucleosides (e.g. α-L-LNA) or a mixture of DNA and nuclease-recruiting nucleosides) composition. In another specific embodiment, the oligonucleotide is a gapmer, wherein each of the F and F'regions is composed of two LNA units, and the region G is composed of 12, 13, and 14 nuclei It is composed of glycoside units, preferably DNA units. Specific gap polymer designs of this nature include 2-12-2, 2-13-2 and 2-14-2. In another specific embodiment, the oligonucleotide is a gapmer, wherein each region of F and F'is independently composed of three LNA units, and region G is composed of 8, 9, 10, 11, It is composed of 12, 13 or 14 nucleoside units, preferably DNA units. Specific gap polymer designs with this property include 3-8-3, 3-9-3, 3-10-3, 3-11-3, 3-12-3, 3-13-3, and 3-14-3 . In another specific embodiment, the oligonucleotide is a gapmer, wherein each region of F and F'consists of 4 LNA units, and region G consists of 8 or 9, 10, 11 or 12. It is composed of a nucleoside unit, preferably a DNA unit. Specific gap polymer designs with this property include 4-8-4, 4-9-4, 4-10-4, 4-11-4, and 4-12-4. The specific gapmer design with this property includes the FG-F' design selected from the group consisting of 1 to 4 modified nucleosides in 6 nucleoside gaps and independent wings, including 1-6-1, 1-6 -2, 2-6-1, 1-6-3, 3-6-1, 1-6-4, 4-6-1, 2-6-2, 2-6-3, 3-6-2 , 2-6-4, 4-6-2, 3-6-3, 3-6-4 and 4-6-3 gapmers. The specific gapmer design with this property includes FG-F' design selected from the group consisting of 1 to 4 modified nucleosides in 7 nucleoside gaps and independent wings, including 1-7-1, 2-7 -1, 1-7-2, 1-7-3, 3-7-1, 1-7-4, 4-7-1, 2-7-2, 2-7-3, 3-7-2 , 2-7-4, 4-7-2, 3-7-3, 3-7-4, 4-7-3 and 4-7-4 gapmers. The specific gapmer design with this property includes FG-F' design selected from the group consisting of 1 to 4 modified nucleosides in 8 nucleoside gaps and independent wings, including 1-8-1, 1-8 -2, 1-8-3, 3-8-1, 1-8-4, 4-8-1, 2-8-1, 2-8-2, 2-8-3, 3-8-2 , 2-8-4, 4-8-2, 3-8-3, 3-8-4, 4-8-3 and 4-8-4 gapmers. The specific gapmer design with this property includes FG-F' design selected from the group consisting of 1 to 4 modified nucleosides in 9 nucleoside gaps and independent wings, including 1-9-1, 2-9 -1, 1-9-2, 1-9-3, 3-9-1, 1-9-4, 4-9-1, 2-9-2, 2-9-3, 3-9-2 , 2-9-4, 4-9-2, 3-9-3, 3-9-4, 4-9-3 and 4-9-4 gapmers. The specific gap polymer design with this property includes the FG-F' design selected from the group consisting of 10 nucleoside gaps, including 1-10-1, 2-10-1, 1-10-2, 1-10-3 , 3-10-1, 1-10-4, 4-10-1, 2-10-2, 2-10-3, 3-10-2, 2-10-4, 4-10-2, 3 -10-3, 3-10-4, 4-10-3 and 4-10-4 gapmers. The specific gapmer design with this property includes the FG-F' design selected from the group consisting of 11 nucleoside gaps, including 1-11-1, 2-11-1, 1-11-2, 1-11-3 , 3-11-1, 1-11-4, 4-11-1, 2-11-2, 2-11-3, 3-11-2, 2-11-4, 4-11-2, 3 -11-3, 3-11-4, 4-11-3 and 4-11-4 gapmers. The specific gap polymer design with this property includes the FG-F' design selected from the group consisting of 12 nucleoside gaps, including 1-12-1, 2-12-1, 1-12-2, 1-12-3 , 3-12-1, 1-12-4, 4-12-1, 2-12-2, 2-12-3, 3-12-2, 2-12-4, 4-12-2, 3 -12-3, 3-12-4, 4-12-3 and 4-12-4 gapmers. The specific gap polymer design with this property includes the FG-F' design selected from the group consisting of 13 nucleoside gaps, including 1-13-1, 2-13-1, 1-13-2, 1-13-3 , 3-13-1, 1-13-4, 4-13-1, 2-13-2, 2-13-3, 3-13-2, 2-13-4, 4-13-2, 3 -13-3, 3-13-4, 4-13-3 and 4-13-4 gapmers. The specific gap polymer design with this property includes FG-F' design selected from the group consisting of 14 nucleoside gaps, including 1-14-1, 2-14-1, 1-14-2, 1-14-3 , 3-14-1, 1-14-4, 4-14-1, 2-14-2, 2-14-3, 3-14-2, 2-14-4, 4-14-2, 3 -14-3, 3-14-4, 4-14-3 and 4-14-4 gapmers. The specific gap polymer design with this property includes the FG-F' design selected from the group consisting of 15 nucleoside gaps, including 1-15-1, 2-15-1, 1-15-2, 1-15-3 , 3-15-1, 1-15-4, 4-15-1, 2-15-2, 2-15-3, 3-15-2, 2-15-4, 4-15-2 and 3 -15-3 gap polymer. The specific gap polymer design with this property includes the FG-F' design selected from the group consisting of 16 nucleoside gaps, including 1-16-1, 2-16-1, 1-16-2, 1-16-3 , 3-16-1, 1-16-4, 4-16-1, 2-16-2, 2-16-3, 3-16-2, 2-16-4, 4-16-2 and 3 -16-3 gap polymer. The specific gap polymer design with this property includes the FG-F' design selected from the group consisting of 17 nucleoside gaps, including 1-17-1, 2-17-1, 1-17-2, 1-17-3 , 3-17-1, 1-17-4, 4-17-1, 2-17-2, 2-17-3 and 3-17-2 gapmers. In all cases, the FG-F' design may further include regions D'and/or D", which may have 1, 2 or 3 nucleoside units (e.g. DNA units, such as 2-phosphodiester-linked DNA units ). Preferably, the nucleosides in regions F and F'are modified nucleosides, and the nucleotides in region G are preferably unmodified nucleosides. In each design, the modified nucleoside is preferably LNA. In another embodiment, all the internucleoside linkages in the gaps in the gapmer are phosphorothioate and/or borane phosphate linkages. In another embodiment, all the internucleoside linkages in the flanks (F and F'regions) in the gapmer are phosphorothioate and/or borane phosphate linkages. In another preferred embodiment, all internucleoside linkages in the D'and D'' regions of the gapmer are phosphodiester linkages. For the specific gapmer as disclosed herein, when the cytosine (C) residue is annotated as 5-methyl-cytosine, in each embodiment, one or more of the cytosine (C) residues are present in the oligonucleotide C may be an unmodified C residue. In a specific embodiment, the so-called short polymer of the interstitial polymerization system is described in WO2008/113832 (which is incorporated herein by reference). Other gapmer designs are disclosed in WO2004/046160, WO2007/146511 and are incorporated herein by reference. For certain embodiments of the present invention, the oligonucleotide is selected from the group of oligonucleotide compounds having CMP-ID-NO: 5_1 to 743_1 and 771_1. For certain embodiments of the present invention, the oligonucleotide is selected from the group of oligonucleotide compounds with the following numbers: CMP-ID-NO 6_1, 8_1, 9_1, 13_1, 41_1, 42_1, 58_1, 77_1 , 92_1, 111_1, 128_1, 151_1, 164_1, 166_1, 169_1, 171_1, 222_1, 233_1, 245_1, 246_1, 250_1, 251_1, 252_1, 256_1, 272_1, 273_1, 287_1, 292_1, 303_1, 314_1, 318_1, 320_1, 324_1 , 336_1, 342_1, 343_1, 344_1, 345_1, 346_1, 349_1, 359_1, 360_1, 374_1, 408_1, 409_1, 415_1, 417_1, 424_1, 429_1, 430_1, 458_1, 464_1, 466_1, 474_1, 490_1, 493_1, 512_1, 519_1 , 519_1, 529_1, 533_1, 534_1, 547_1, 566_1, 567_1, 578_1, 582_1, 601_1, 619_1, 620_1, 636_1, 637_1, 638_1, 640_1, 645_1, 650_1, 651_1, 652_1, 653_1, 658_1, 659_1, 660_1, 665_1 , 678_1, 679_1, 680_1, 682_1, 683_1, 684_1, 687_1, 694_1, 706_1, 716_1, 728_1, 733_1, 734_1, and 735_1. In a preferred embodiment of the present invention, the oligonucleotide is CMP-ID-NO: 287_1. In another preferred embodiment of the present invention, the oligonucleotide is CMP-ID-NO: 342_1. In another preferred embodiment of the present invention, the oligonucleotide is CMP-ID-NO: 640_1. In another preferred embodiment of the present invention, the oligonucleotide is CMP-ID-NO: 466_1. In another preferred embodiment of the present invention, the oligonucleotide is CMP-ID-NO: 566_1. In another embodiment of the present invention, the adjacent nucleotide sequence of the oligonucleotide motif and oligonucleotide compound of the present invention includes 2 to 4 other phosphodiesters at the 5'end of the adjacent nucleotide sequence Linked nucleosides (for example, region D'). In one example, these nucleosides are used as biocleavable linkers (see the section on biocleavable linkers). In a preferred embodiment, the ca (cytidine-adenosine) dinucleotide is linked to the adjacent nucleotide sequence (that is, the motif sequence or the oligonucleotide compound listed in Table 5) via a phosphodiester link Any one of them) at the 5'end. In a preferred embodiment, the ca dinucleotide is not complementary to the target sequence where the remaining part of the adjacent nucleotide is complementary. In some embodiments of the present invention, the oligonucleotide or adjacent nucleotide sequence is selected from the group consisting of nucleotide motif sequences having SEQ ID NOs: 766, 767, 768, 769, and 770. In some embodiments of the present invention, the oligonucleotide is selected from the group consisting of oligonucleotide compounds having CMP-ID-NO 766_1, 767_1, 768_1, 769_1 and 770_1.Carbohydrate conjugate portion The carbohydrate conjugate portion includes, but is not limited to, galactose, lactose, n-acetylgalactosamine, mannose, and mannose-6-phosphate. Carbohydrate conjugates can be used to enhance delivery or activity in a variety of tissues, such as liver and/or muscle. For example, see EP1495769, WO99/65925, Yang et al., Bioconjug Chem (2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10): 1401-17. In some embodiments, the carbohydrate conjugate moiety is multivalent, for example, 2, 3 or 4 identical or different carbohydrate moieties can be covalently joined to the oligonuclear via one or more linkers as appropriate. Glycidic acid. In some embodiments, the present invention provides conjugates that include the oligonucleotide and carbohydrate conjugate portions of the present invention. In some embodiments, the conjugate moiety may include mannose or mannose-6-phosphate. This is particularly useful for targeting muscle cells, see for example US 2012/122801. The conjugate moiety capable of binding to the asialoglycoprotein receptor (ASGPr) is particularly useful for targeting hepatocytes in the liver. In some embodiments, the present invention provides oligonucleotide conjugates that include the oligonucleotides of the present invention and the conjugate portion targeting the asialoglycoprotein receptor. The conjugate moiety that targets the asialoglucoprotein receptor includes one or more carbohydrate moieties (the carbohydrate moiety that binds to ASPGr) that can bind to the asialoglucoprotein receptor with an affinity equal to or greater than galactose. The affinity of many galactose derivatives to asialoglycoprotein receptors has been studied (see, for example: Jobst, S.T. and Drickamer, K. JB.C. 1996, 271, 6686) or can be easily measured using typical methods in the industry. One aspect of the present invention includes the following antisense oligonucleotide conjugates: a) Oligonucleotide (region A), which includes a length of 10 to 30 that has at least 90% complementarity with the PD-L1 target nucleic acid A contiguous nucleotide sequence of three nucleotides; and b) at least one conjugate portion (region C) targeting the asialoglycoprotein receptor, which is covalently linked to the oligonucleotide in a). The oligonucleotide or contiguous nucleotide sequence may be as described in any of the "oligonucleotides of the present invention", "oligonucleotide design" and "gapmer design". In some embodiments, the conjugate moiety targeting the asialoglycoprotein receptor includes at least one ASPGr-binding carbohydrate moiety selected from the group consisting of galactose, galactosamine, N-methanyl- Galactosamine, N-acetylgalactosamine, N-propylgalactosamine, N-butyryl-galactosamine and N-isobutyrylgalactosamine. In some embodiments, the conjugate moiety that targets the asialoglycoprotein receptor is monovalent, bivalent, trivalent, or tetravalent (that is, contains 1, 2, 3, or 4 conjugates capable of binding to the asialoglycoprotein The terminal carbohydrate portion of the protein receptor). Preferably, the part of the conjugate targeted to the asialoglycoprotein receptor is bivalent, even more preferably trivalent. In a preferred embodiment, the conjugate portion targeting the asialoglycoprotein receptor includes 1 to 3 N-acetylgalactosamine (GalNAc) moieties (also known as GalNAc conjugates). In some embodiments, the oligonucleotide coupling includes the conjugate portion of the trivalent N-acetylgalactosamine (GalNAc) moiety that targets the asialoglycoprotein receptor. GalNAc conjugates have been combined with phosphodiester, methylphosphonate and PNA antisense oligonucleotides (such as US 5,994517 and Hangeland et al., Bioconjug Chem. 1995 Nov-Dec; 6(6):695-701 ; Biessen et al., 1999 Biochem J. 340, 783-792 and Maier et al., 2003 Bioconjug Chem 14, 18-29) and siRNA (such as WO 2009/126933, WO 2012/089352 and WO 2012/083046) and LNA and 2'-MOE modified nucleosides (WO 2014/076196, WO 2014/207232 and WO 2014/179620 (incorporated herein by reference)) are used together. To generate the conjugate moiety that targets the asialoglycoprotein receptor, the ASPGr-binding carbohydrate moiety (preferably GalNAc) is connected to the branching agent molecule via the C-1 carbon of the sugar. The ASPGr binding carbohydrate moiety is preferably connected to the branching agent molecule via a spacer. The preferred spacer system is a flexible hydrophilic spacer (US Patent 5,885,968; Biessen et al. J. Med. Chern. 1995, Vol. 39, pp. 1538-1546). Preferred is a flexible hydrophilic spacer system PEG spacer. The preferred PEG spacer system is PEG3 spacer (three ethylene units). The branching agent molecule can be any small molecule that allows the connection of two or three ASPGr-binding terminal carbohydrate moieties and further allows the branch point to be connected to the oligonucleotide. An exemplary branching agent molecule is dilysine. The dilysine molecule contains three amine groups (through which three carbohydrate moieties that bind ASPGr can be connected) and a carboxy-reactive group (through which the dilysine can be connected to the oligonucleotide). The alternative branching agent molecule can be a diploid or a triploid, such as those supplied by Glen Research. In some embodiments, the branching agent may be selected from the group consisting of: 1,3-bis-[5-(4,4'-dimethoxytrityloxy)pentylamido]propane 2-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (Glen Research catalog number: 10-1920-xx), reference-2,2,2- [3-(4,4'-Dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl) ]-Phosphoramidite (Glen Research catalog number: 10-1922-xx), reference-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyl oxygen Methyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite and 1-[5-(4,4'- Dimethoxy-trityloxy)pentylamino]-3-[5-茀methoxy-carbonyl-oxy-pentylamino]-propyl-2-[(2- Cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research catalog number: 10-1925-xx). WO 2014/179620 and PCT Application No. PCT/EP2015/073331 describe the generation of various GalNAc conjugate moieties (incorporated herein by reference). One or more linkers can be inserted between the branching agent molecule and the oligonucleotide. In a preferred embodiment, the linking system is bio-cleavable linker. The linker can be selected from the linkers described in the "linker" part and its subparts. The method known in the art can be used to link the conjugate portion, especially the GalNAc conjugate portion, that targets the asialoglycoprotein receptor to the 3'-end or 5'-end of the oligonucleotide. In a preferred embodiment, the conjugate moiety targeting the asialoglycoprotein receptor is attached to the 5'-end of the oligonucleotide. Pharmacokinetic modulators related to siRNA delivery have been described in WO2012/083046 (incorporated herein by reference). In some embodiments, the carbohydrate conjugate portion includes a pharmacokinetic modifier selected from the group consisting of: a hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16-20 carbon atoms Groups, palmitoyl, hexadecenyl, oleyl, (9E,12E)-octadecenyl-9,12 dienyl, dioctyl and C16-C20 acyl and cholesterol. In a preferred embodiment, the portion of the carbohydrate conjugate containing the pharmacokinetic modifier is a GalNAc conjugate. Preferred carbohydrate conjugate moieties include one to three ASPGr-bound terminal carbohydrate moieties, preferably N-acetylgalactosamine moieties. In some embodiments, the carbohydrate conjugate moiety includes three ASPGr-bound carbohydrate moieties, preferably N-acetylgalactosamine moieties, connected to the branching agent molecule via a spacer. Spacer molecules can be 8 to 30 atoms in length. The preferred carbohydrate conjugate moiety includes three terminal GalNAc moieties connected to the dilysine brancher molecule via a PEG spacer. Preferably, the PEG spacer system is 3PEG spacer. The portion of the conjugate suitable for targeting the asialoglycoprotein receptor is shown in FIG. 1. The portion of the conjugate that preferably targets the asialoglycoprotein receptor is shown in FIG. 3. Other GalNAc conjugate moieties may include, for example, small peptides linked to GalNAc moieties, such as Tyr-Glu-Glu-(aminohexylGalNAc)3 (YEE(ahGalNAc)3; asialoglycans bound to hepatocytes Glycotripeptides of protein receptors, for example, see Duff et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose clusters (for example, L3G4; Biessen et al., Cardovasc. Med., 1999, 214); And cholane-based galactose clusters (such as carbohydrate recognition motifs for asialoglycoprotein receptors). In some embodiments of the present invention, the antisense oligonucleotide conjugate is selected from the group consisting of CPM ID NO: 766_2, 767_2, 768_2, 769_2, and 770_2. In a preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound shown in FIG. 4. In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound shown in FIG. 5. In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound shown in FIG. 6. In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound shown in FIG. 7. In another preferred embodiment, the antisense oligonucleotide conjugate corresponds to the compound shown in FIG. 8.Connector Biocleavable linker ( area B) The use of conjugates generally enhances pharmacokinetic or pharmacodynamic properties. However, the presence of the conjugate moiety can, for example, interfere with the activity of the oligonucleotide against its intended target via a steric hindrance that hinders hybridization or nuclease recruitment (e.g., RNAseH). The use of a physiologically unstable bond (biocleavable linker) between the oligonucleotide (region A or the first region) and the conjugate part (region C or the third region) allows for improvement due to the presence of the conjugate part Nature, while ensuring that the coupling group at the target tissue does not interfere with the effective activity of the oligonucleotide. When molecules containing physiologically unstable bonds reach the appropriate intracellular and/or extracellular environment, the unstable bonds are spontaneously cleaved. For example, when a molecule enters an acidified endosome, the pH-labile bond can be cleaved. Therefore, the pH-labile bond can be regarded as an endosomal cleavable bond. Enzyme-cleavable bonds can be cleaved upon exposure to enzymes, such as those present in endosomes or lysosomes or in the cytoplasm. Disulfide bonds can be cleaved when molecules enter the higher reducing environment of the cytoplasm. Therefore, disulfides can be regarded as cytoplasmic cleavable bonds. As used herein, pH labile bonds are labile bonds that are selectively broken under acidic conditions (pH<7). These bonds can also be called endosomal unstable bonds, because the pH of the cell endosomes and lysosomes is less than 7. For the biocleavable linker associated with the conjugate moiety for targeted delivery, preferably, the lysis rate seen in the target tissue (such as muscle, liver, kidney or tumor) is greater than that found in serum In the middle. Suitable methods for determining the degree of lysis (%) of target tissue and serum or S1 nuclease cleavage are described in the "Materials and Methods" section. In some embodiments, the biocleavable linker (also known as physiologically unstable linker or nuclease susceptible linker or region B) in the conjugate of the present invention is cleaved by at least about 20% compared to the standard, for example At least about 30% lysis, such as at least about 40% lysis, such as at least about 50% lysis, such as at least about 60% lysis, such as at least about 70% lysis, such as at least about 75% lysis. In some embodiments, the oligonucleotide conjugate of the present invention includes three regions: i) the first region (region A), which includes 10-25 adjacent nucleotides complementary to the target nucleic acid; ii) the first region (region A); The second region (region B), which includes a biocleavable linker; and iii) the third region (region C), which includes a conjugate portion, such as a conjugate portion targeting an asialoglycoprotein receptor, wherein The third region is covalently connected to the second region that is covalently connected to the first region. In an embodiment of the present invention, the oligonucleotide conjugate includes a bioavailable portion between the adjacent nucleotide sequence (region A) and the conjugate portion (region C) targeting the asialoglycoprotein receptor. Cleavage of the linker (region B). In some embodiments, the biocleavable linker can be located at the 5'end and/or 3'end of the adjacent nucleotide (region A) complementary to the target nucleic acid. In a preferred embodiment, the biocleavable linker is located at the 5'end. In some embodiments, the cleavable linker is susceptible to nucleases that can, for example, be expressed in target cells. In some embodiments, the biocleavable linkage system consists of 2 to 5 consecutive phosphodiester linkages. The linker can be a short region (e.g., 1-10, as detailed in the linker definition) nucleoside linked by a phosphodiester. In some embodiments, the nucleoside (independently) in the biocleavable linker region B is selected from the group consisting of DNA and RNA or modifications that do not interfere with nuclease cleavage. DNA and RNA nucleoside modifications that do not interfere with nuclease cleavage can be unnatural nucleobases. Certain sugar-modified nucleosides can also allow nuclease cleavage, such as α-L-oxy-LNA. In some embodiments, all nucleosides in region B include (as appropriate independently) 2'-OH ribose sugar (RNA) or 2'-H sugar-i.e. RNA or DNA. In a preferred embodiment, at least two consecutive nucleosides in region B are DNA or RNA nucleosides (for example, at least 3 or 4 or 5 consecutive DNA or RNA nucleosides). In an even more preferred embodiment, the nucleoside of region B is a DNA nucleoside. Preferably, region B is composed of 1 to 5 or 1 to 4 (for example, 2, 3, 4) consecutive phosphodiester-linked DNA nucleosides. In a preferred embodiment, region B is shorter so that it does not recruit RNaseH. In some embodiments, region B includes no more than 3 or no more than 4 consecutive phosphodiester-linked DNA and/or RNA nucleosides (eg, DNA nucleosides). In the case where region B is composed of nucleosides linked by phosphodiester, regions A and B can form an oligonucleotide linked to region C together. In this context, the difference between region A and region B may be that region A starts with at least one, preferably at least two modified nucleosides (such as LNA or with 2' Substitute the nucleoside of the sugar moiety) and region A itself can regulate the performance of the target nucleic acid in the relevant cell line. In addition, if region A includes DNA or RNA nucleosides, these nucleosides are linked to nuclease-resistant nucleosides (for example, phosphorothioate or borane phosphate). On the other hand, region B includes a phosphodiester link between DNA and RNA nucleosides. In some embodiments, region B is not complementary to the target nucleic acid or includes at least a 50% mismatch with respect to the target nucleic acid. In some embodiments, region B is not complementary to the target nucleic acid sequence or adjacent nucleotides complementary to the target nucleic acid in region A. In some embodiments, region B is complementary to the target nucleic acid sequence. In this regard, regions A and B together can form a single contiguous sequence complementary to the target sequence. In some aspects of the present invention, the internucleoside link between the first region (region A) and the second region (region B) can be regarded as a part of the second region. In some embodiments, the base sequence in region B is selected to provide the best endonuclease cleavage site based on the major endonuclease cleavage enzymes present in the target tissue or cell or sub-cell compartment. In this regard, by separating cell extracts from target tissues and non-target tissues, the area can be selected based on the preferential lysis activity in the desired target cells (eg liver/hepatocytes) compared to non-target cells (eg kidney) Endonuclease cleavage sequence in B. In this regard, the efficacy of the compound on target down-regulation can be optimized for the desired tissue/cell. In some embodiments, region B includes two nucleotides of the sequence AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, where C can be It is 5-methylcytosine, and/or T can be replaced by U. Preferably, the internucleoside linkage is a phosphodiester linkage. In some embodiments, region B includes the sequence AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, Three nucleotides of CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC and GGG, where C can be 5-A The cytosine and/or T can be replaced by U. Preferably, the internucleoside linkage is a phosphodiester linkage. In some embodiments, region B includes the sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX, CCGX, CGAX, Trinucleotides of CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX and GGGX, where X can be selected from the following components The group: A, T, U, G, C and the like, where C can be 5-methylcytosine and/or T can be replaced by U. Preferably, the internucleoside linkage is a phosphodiester linkage. It should be recognized that when referring to (natural) nucleobases A, T, U, G, C, these nucleobases can be used as nucleobase analogs of equivalent natural nucleobases (for example, with complementary nucleobases). The base pair of the glycoside) is substituted.Other connectors ( area Y) The linker may have at least two functional groups, one functional group for attachment to the oligonucleotide and the other for attachment to the conjugate moiety. Exemplary linker functional groups can be electrophilic for reacting with nucleophilic groups on the oligonucleotide or conjugate moiety, or nucleophilic for reacting with electrophilic groups . In some embodiments, the linker functional group includes an amine group, a hydroxyl group, a carboxylic acid, a thiol, an amino phosphate, a phosphorothioate, a phosphate, a phosphite, an unsaturated group (such as a double bond or a triple bond). ) And so on. Some example linkers (region Y) include 8-amino-3,6-dioxaoctanoic acid (ADO), 4-(N-maleimidinylmethyl)cyclohexane-1-carboxylic acid succinate Imino ester (SMCC), 6-aminohexanoic acid (AHEX or AHA), 6-aminohexyloxy, 4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl 4- (N-maleiminomethyl) cyclohexane-l-carboxy-(6-aminohexanoate) (LCSMCC), m-maleimino-benzoic acid succinimidyl Ester (MBS), Ne-maleimino-octanoic acid succinimidyl ester (EMCS), 6-(β-maleimino-propionamido) hexanoic acid succinimidyl ester (SMPH), succinimidyl N-(a-maleimidinyl acetate) (AMAS), 4-(p-maleimidinylphenyl) butyric acid succinimidyl ester (SMPB), β-alanine (β-ALA), phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC), β-(cyclopropyl)alanine (β-CYPR), amine Dodecanoic acid (ADC), alkylene glycol, polyethylene glycol, amino acid and the like. In some embodiments, the linker (region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group (including, for example, a C6-C12 aminoalkyl group). In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group. The amino alkyl amino phosphate amino ester alkyl ester can be added to the oligonucleotide (region A or region A-B) as part of standard oligonucleotide synthesis, for example, using (e.g. protected) aminoalkylamino phosphate amino ester alkyl esters. The linking group between the aminoalkyl group and the oligonucleotide can be, for example, phosphorothioate or phosphodiester or one of the other nucleoside linking groups mentioned herein. The aminoalkyl group is covalently attached to the 5'or 3'end of the oligonucleotide. Commercially available aminoalkyl linking systems (for example) 3'-amine-modifier reagents (for linking at the 3'end of oligonucleotides), and for linking at the 5'end of oligonucleotides In other words, 5'-amino-modifier C6 can be used. These reagents are available from Glen Research Corporation (Sterling, Va.). These compounds or analogs were used in Krieg et al., Antisense Research and Development 1991, 1, 161 to attach Lucifer Yellow to the 5'-end of oligonucleotides. Numerous other linker groups are known in the industry and can be used to link conjugate moieties to oligonucleotides. A review of many useful linker groups can be found, for example, in Antisense Research and Applications, edited by S. T. Crooke and B. Lebleu, CRC Press, Boca Raton, Fla., 1993, pages 303-350. Other compounds, such as acridine, are linked to the 3'-terminal phosphate group of the oligonucleotide via a polymethylene link (Asseline et al., Proc. Natl. Acad. Sci. USA 1984, 81, 3297). Any of the above groups can be used as a single linker (region Y) or in combination with one or more other linkers (region Y-Y' or region Y-B or B-Y). Linkers and their use in preparing oligonucleotide conjugates have been widely provided in the industry, such as WO 96/11205 and WO 98/52614 and US Pat. No. 4,948,882; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025; 5,254,469; 5,245,022; 5,112,963; 5,391,723; 5,510475; 5,512,667; 5,574,142; 5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437 of each of these cases Incorporated into this article by reference. Manufacturing method In another aspect, the present invention provides a method of manufacturing the oligonucleotide of the present invention, which comprises reacting the nucleotide unit and thereby forming a covalently connected neighbor included in the oligonucleotide Nucleotide unit. Preferably, the method uses phosphoramidite chemistry (see, for example, Caruthers et al., 1987, Methods in Enzymology, Vol. 154, pages 287-313). In another embodiment, the method further includes reacting the adjacent nucleotide sequence with the conjugate moiety (ligand). In another aspect, a method for manufacturing the composition of the present invention is provided, which comprises mixing the oligonucleotide or coupling oligonucleotide of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt and/ Or adjuvant. Pharmaceutical composition In another aspect, the present invention provides a pharmaceutical composition, which includes any of the above-mentioned oligonucleotides and/or oligonucleotide conjugates and pharmaceutically acceptable diluents, Solvents, carriers, salts and/or adjuvants. The pharmaceutically acceptable diluent includes phosphate buffered saline (PBS) and the pharmaceutically acceptable salt includes, but is not limited to, sodium salt and potassium salt. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the oligonucleotide is used in a pharmaceutically acceptable diluent at a concentration of 50-300 µM solution. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990). WO2007/031091 provides other suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Suitable dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO2007/031091. The oligonucleotides or oligonucleotide conjugates of the present invention can be mixed with pharmaceutically acceptable active or inert substances to prepare pharmaceutical compositions or formulations. The composition and the method of formulating the pharmaceutical composition depend on many criteria, including (but not limited to) the route of administration, the degree of disease, or the dose to be administered. These compositions can be sterilized by conventional sterilization techniques, or can be sterile filtered. The resulting aqueous solution can be packaged for use as it is or lyophilized, and the lyophilized formulation can be combined with a sterile aqueous vehicle before administration. The pH of the formulation is usually between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8 (for example, 7 to 7.5). The resulting composition in solid form can be packaged into a plurality of single dosage units, each unit containing a fixed amount of one or more of the above-mentioned agents, for example in the form of a sealed package of tablets or capsules. The composition in solid form can also be packaged into a container for flexible quantities, for example in the form of a squeezable tube designed for topical application of creams or ointments. In some embodiments, the oligonucleotides or oligonucleotide conjugates of the present invention are prodrugs. In particular, for oligonucleotide conjugates, after delivery of the prodrug to the site of action (eg, target cells), the conjugate portion is immediately separated from the oligonucleotide. Application The oligonucleotides or oligonucleotide conjugates of the present invention can be used as research reagents for diagnosis, treatment and prevention, for example. In research, these oligonucleotides or oligonucleotide conjugates can be used to specifically regulate the synthesis of PD-L1 protein in cells (such as in vitro cell cultures) and experimental animals, thereby promoting target Functional analysis or evaluation of its usefulness as a target for therapeutic intervention. Generally, target regulation is achieved by degrading or inhibiting protein-producing mRNA (thus preventing the formation of proteins) or by degrading or inhibiting protein-producing genes or modulators of mRNA. If the oligonucleotide of the present invention is used in research or diagnosis, the target nucleic acid can be cDNA or a synthetic nucleic acid derived from DNA or RNA. The present invention provides an in vivo or in vitro method for modulating PD-L1 expression in a target cell expressing PD-L1, the method comprising administering to the cell an effective amount of the oligonucleotide or oligonucleotide coupling of the present invention Things. In some embodiments, the target cell line is a mammalian cell, especially a human cell. The target cell can be an in vitro cell culture or an in vivo cell forming part of a tissue in a mammal. In a preferred embodiment, the target cell is present in the liver. Hepatic target cells can be selected from parenchymal cells (e.g., hepatocytes) and non-parenchymal cells (e.g., Kuffard cells, LSEC, astrocytes (or Ito cells), bile duct epithelial cells, and liver-related white blood cells (including T cells and NK cells) ). In some embodiments, the target cell line is an antigen presenting cell. Antigen-presenting cells show foreign antigens in complex with the class I or class II major histocompatibility complex (MHC) on their surface. In some embodiments, the antigen-presenting cells exhibit class II MHC (ie, occupational antigen-presenting cells, such as dendritic cells, macrophages, and B cells). In diagnosis, oligonucleotides can be used to detect and quantify PD-L1 expression in cells and tissues by northern blotting (northern blotting), in situ hybridization or similar techniques. For treatment, the oligonucleotide or oligonucleotide conjugate of the present invention or the pharmaceutical composition thereof can be administered to an animal or human suspected of having a disease or condition, and the disease or condition can be reduced by PD- L1 performance, especially by reducing PD-L1 performance in liver target cells, can be alleviated or treated. The present invention provides a method for treating or preventing a disease, which comprises administering a therapeutically or preventively effective amount of the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention to an individual suffering from or susceptible to the disease. The present invention also relates to the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention for use as a medicament. The oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention is usually administered in an effective amount. The present invention also provides the use of the described oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention to manufacture medicaments for the treatment of diseases or disorders as mentioned herein. In one embodiment, the disease is selected from a) viral liver infections, such as HBV, HCV and HDV; b) parasitic infections, such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; and c) Liver cancer or metastasis in the liver. In one embodiment, the present invention relates to oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions for the treatment of diseases or conditions selected from viral or parasitic infections. In another embodiment, the disease is selected from a) viral liver infections such as HBV, HCV and HDV; b) parasitic infections such as malaria, toxoplasmosis, leishmaniasis and trypanosomiasis; and c ) Liver cancer or metastasis in the liver. The diseases or conditions mentioned herein are related to immune depletion. In particular, the disease or condition is related to the exhaustion of the virus-specific T cell response. In some embodiments, the reduction of PD-L1 performance can be used to alleviate or treat a disease or condition. The method of the present invention is preferably used to treat or prevent diseases related to immune depletion. In one embodiment of the present invention, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention can be used to restore the immune response against liver cancer or metastasis in the liver. In one embodiment of the present invention, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention can be used to restore the immune response against pathogens. In some embodiments, pathogens can be found in the liver. The pathogen may be a virus or a parasite, especially those described herein. In a preferred embodiment, the pathogenic system is HBV. The present invention also relates to the use of oligonucleotides, oligonucleotide conjugates or pharmaceutical compositions as defined herein, which are used to manufacture for the restoration of immunity against viral or parasitic infections as mentioned herein的药。 The medicine. The oligonucleotide or oligonucleotide conjugate or pharmaceutical composition of the present invention can be used to treat viral infections, especially viral infections in the liver that affect the PD-1 pathway (for example, see Kapoor and Kottilil 2014 Future Virol Vol. 9 No. 9 565-585 pages and Salem and El-Badawy 2015 World J Hepatol Volume 7, pages 2449-2458). Viral liver infections can be selected from the group consisting of hepatitis viruses, especially HBV, HCV and HDV, especially chronic forms of these infections. In one embodiment, the oligonucleotide or oligonucleotide conjugate or pharmaceutical composition of the present invention is used to treat HBV, especially chronic HBV. The indicators of chronic HBV infection are high circulating viral load (HBV DNA) and even higher levels of empty HBsAg particles (>100-fold excess virions). The oligonucleotides or oligonucleotide conjugates of the present invention can also be used to treat viral liver infections co-infected with HIV. Other viral infections that can be treated with the oligonucleotide or oligonucleotide conjugate or pharmaceutical composition of the present invention are lcmv (lymphocytic choroid meningitis virus) and HIV (in the form of a single bacterial infection), HSV -1 and HSV-2 and other herpes viruses. These viruses are not prone to infect hepatocytes, however, they can be sensitive to down-regulation of PDL1. In some embodiments, restoration of immunity or immune response involves improving T cell and/or NK cell response and/or alleviating T cell exhaustion, in particular, restoring HBV-specific T cell response, HCV-specific T cell response, and Or HDV-specific T cell response. The improvement in T cell response can be evaluated, for example, as an increase in T cells in the liver, especially an increase in CD8+ and/or CD4+ T cells compared to a control (e.g., a value before treatment or a value in a vehicle-treated individual). In another embodiment, the virus-specific CD8+ T cells are recovered or increased compared to the control. Specifically, HBV-specific CD8+ T cells or HCV-specific CD8+ T cells or HDV-specific CD8+ T cells are compared with the control. The ratio has recovered or increased. In a preferred embodiment, in an individual treated with the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention, compared with a control, HBV s antigen (HBsAg) specific CD8+ T cells And/or HBV e antigen (HBeAg) specific CD8+ T cells and/or HBV core antigen (HBcAg) specific CD8+ T cells increased. Preferably, HBV antigen-specific CD8+ T cells produce one or more cytokines (for example, interferon-γ (IFN-γ) or tumor necrosis factor α (TNF-α)). In particular, an increase in the above-mentioned CD8+ T cells was observed in the liver. When compared with the control, the increase described in this article should be statistically significant. Preferably, the increase is at least 20% (e.g. 25%, e.g. 50%, e.g. 75%) when compared to the control. In another embodiment, natural killer (NK) cells and/or natural killer T (NKT) cells are activated by the oligonucleotide or oligonucleotide conjugate of the present invention. The oligonucleotides or oligonucleotide conjugates or pharmaceutical compositions of the present invention can be used to treat parasitic infections, especially parasitic infections affecting the PD-1 pathway (for example, see Bhadra et al., 2012 J Infect Dis Vol. 206 Pages 125-134; Bhadra et al., 2011 Proc Natl Acad Sci USA, Vol. 108, pp. 9196-9201; Esch et al., J Immunol, Vol. 191, pp. 5542-5550; Freeman and Sharpe 2012 Nat Immunol, Vol. 13, p. Pages 113-115; Gutierrez et al., 2011 Infect Immun Volume 79, pages 1873-1881; Joshi et al., 2009 PLoS Pathog Volume 5 e1000431; Liang et al., 2006 Eur J Immunol Volume 36, pages 58-64; Wykes et al., 2014 Front Microbiol Vol. 5, p. 249). Parasitic infections can be selected from the group consisting of malaria, toxoplasmosis, leishmaniasis and trypanosomiasis. Malaria infection is caused by the protozoa of the genus Plasmodium, especially the species Plasmodium vivax, Plasmodium vivax and Plasmodium falciparum. Toxoplasmosis is caused by Toxoplasma (Toxoplasma gondii ) Caused by parasitic diseases. Leishmaniasis is caused by Leishmania (Leishmania ) Diseases caused by protozoan parasites. Trypanosomiasis is caused by Trypanosoma brucei (Trypanosoma ) Caused by protozoa of the genus. Chaga disease is caused by the species Trypanosoma cruzi (Trypanosoma cruzi ) Caused by the tropical form, and the sleep disease is caused by the species Trypanosoma brucei (Trypanosoma brucei )cause. In some embodiments, the restoration of immunity involves the restoration of parasite-specific T cell and NK cell responses, especially Plasmodium-specific T cell responses, Toxoplasma-specific T cell and NK cell responses, and Leishmania-specific Sex T cell and NK cell response, Trypanosoma cruzi-specific T cell and NK cell response, or Trypanosoma brucella-specific T cell and NK cell response. In another embodiment, the parasite-specific CD8+ T cell and NK cell responses are restored. Administration can be local (e.g. skin, inhalation, eyes or ears) or enteral (e.g. oral or gastrointestinal) or parenteral (e.g. intravenous, subcutaneous, intramuscular, intracerebral, intracerebral ventricle or sheath Inner) administer the oligonucleotide or pharmaceutical composition of the present invention. In a preferred embodiment, by parenteral route (including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial (such as intracerebral) or intraventricular, intravitreal) Administration) Administration of the oligonucleotide or pharmaceutical composition of the present invention. In one embodiment, the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment, the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously. In some embodiments, 0.1-15 mg/kg (such as 0.1-10 mg/kg, such as 0.2-10 mg/kg, such as 0.25-10 mg/kg, such as 0.1-5 mg/kg, such as 0.2-5 mg/kg, for example, 0.25-5 mg/kg) is administered to the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention. It can be administered once a week, every second week, every third week, or even once a month. Combination therapy In some embodiments, the oligonucleotide, oligonucleotide conjugate or pharmaceutical composition of the present invention is used in combination therapy with another therapeutic agent. The therapeutic agent may be, for example, standard care for the aforementioned diseases or conditions. To treat chronic HBV infection, it is recommended to use a combination of antiviral drugs and immune system modulators as standard care. Antiviral drugs that are effective against HBV are, for example, nucleoside (acid) analogs. Five nucleoside (acid) analogues have been approved for the treatment of HBV, namely lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread) , Telbivudine (Tyzeka), entecavir (Baraclude), these drugs effectively inhibit viral replication (HBV DNA), but have no effect on HBsAg content. Other antiviral drugs include ribavirin and HBV antibody therapy (single strain or multiple strains). Immune system modulators can be, for example, interferon alpha-2a and pegylated interferon alpha-2a (Pegasys) or TLR7 agonists (e.g. GS-9620) or therapeutic vaccines. IFN-α treatment only showed a very slight effect of reducing the viral load, but produced a certain degree of HBsAg reduction, but the reduction was extremely low (<10% after 48 weeks of therapy). The oligonucleotide or oligonucleotide conjugate of the present invention can also be combined with other antiviral drugs that are effective against HBV (for example, the antisense oligonucleotides described in WO2012/145697 and WO 2014/179629 or described in WO 2005/014806, WO 2012/024170, WO 2012/2055362, WO 2013/003520 and WO 2013/159109 siRNA molecules). When administering the oligonucleotide or oligonucleotide conjugate of the present invention and other agents in combination therapy, they can be administered to the individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the present invention may include the oligonucleotide or oligonucleotide conjugate of the present invention and a pharmaceutically acceptable excipient as described herein and another therapeutic or prophylactic agent known in the industry The combination. Embodiments The following embodiments of the present invention can be used in combination with any other embodiments described herein. 1. An antisense oligonucleotide comprising or consisting of a contiguous nucleotide sequence of 10 to 30 nucleotides in length and capable of reducing PD-L1 performance. 2. The oligonucleotide of embodiment 1, wherein the adjacent nucleotide sequence is at least 90% complementary to the PD-L1 target nucleic acid. 3. The oligonucleotide of embodiment 1 or 2, wherein the adjacent nucleotide sequence is complementary to a target nucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO : 3. 4. The oligonucleotides of Examples 1 to 3, wherein the adjacent nucleotide sequence is complementary to the regions within positions 1 and 15720 of SEQ ID NO:1. 5. The oligonucleotide of embodiments 1 to 4, wherein the oligonucleotide can hybridize to a target nucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID with a ΔG° lower than -10 kcal NO: 2 and/or SEQ ID NO: 3. 6. The oligonucleotide of embodiments 1 to 5, wherein the adjacent nucleotide sequence is complementary to a subsequence of the target nucleic acid, wherein the subsequence is selected from the group consisting of: position 371 on SEQ ID NO: 1 To 3068, 5467 to 12107, 15317 to 15720, 15317-18083, 15317 to 19511, and 18881 to 19494. 7. The oligonucleotide of embodiment 6, wherein the subsequence is selected from the group consisting of: positions 7300 to 7333, 8028 to 8072, 9812 to 9859, 11787 to 11873, and 15690 to positions on SEQ ID NO: 1. 15735. 8. The oligonucleotide of embodiment 2 to 7, wherein the target nucleic acid is RNA. 9. The oligonucleotide of embodiment 8, wherein the RNA is mRNA. 10. The oligonucleotide of embodiment 9, wherein the mRNA is an mRNA precursor or mature mRNA. 11. The oligonucleotide of embodiments 1 to 10, wherein the contiguous nucleotide sequence includes at least 14 contiguous nucleotides, especially 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 Contiguous nucleotides or consist of them. 12. The oligonucleotide of embodiments 1 to 10, wherein the contiguous nucleotide sequence includes or consists of 16 to 20 nucleotides. 13. The oligonucleotide of embodiments 1 to 10, wherein the oligonucleotide includes or consists of 14 to 35 nucleotides (length). 14. The oligonucleotide of embodiment 13, wherein the oligonucleotide includes or consists of 18 to 22 nucleotides (length). 15. The oligonucleotide of embodiments 1 to 14, wherein the oligonucleotide or adjacent nucleotide sequence is single-stranded. 16. The oligonucleotide of embodiments 1 to 15, wherein the adjacent nucleotide sequence is complementary to a subsequence of the target nucleic acid, wherein the subsequence is selected from the group consisting of: A7, A26, A43, A119, A142 , A159, A160, A163, A169, A178, A179, A180, A189, A201, A202, A204, A214, A221, A224, A226, A243, A254, A258, 269, A274, A350, A360, A364, A365, A370 , A372, A381, A383, A386, A389, A400, A427, A435 and A438. 17. The oligonucleotide of embodiment 16, wherein the subsequence is selected from the group consisting of A221, A360, A180, A160 and A269. 18. The oligonucleotide of Examples 1 to 17, wherein the oligonucleotide is not siRNA and is not self-complementary. 19. The oligonucleotide of embodiments 1 to 18, wherein the contiguous nucleotide sequence includes or consists of a sequence selected from SEQ ID NO: 5 to 743 or 771. 20. The oligonucleotide of embodiments 1 to 19, wherein the contiguous nucleotide sequence includes or consists of a sequence selected from: SEQ ID NO: 6, 8, 9, 13, 41, 42, 58, 77, 92, 111, 128, 151, 164, 166, 169, 171, 222, 233, 245, 246, 250, 251, 252, 256, 272, 273, 287, 292, 303, 314, 318, 320, 324, 336, 342, 343, 344, 345, 346, 349, 359, 360, 374, 408, 409, 415, 417, 424, 429, 430, 458, 464, 466, 474, 490, 493, 512, 519, 519, 529, 533, 534, 547, 566, 567, 578, 582, 601, 619, 620, 636, 637, 638, 640, 645, 650, 651, 652, 653, 658, 659, 660, 665, 678, 679, 680, 682, 683, 684, 687, 694, 706, 716, 728, 733, 734 and 735. 21. The oligonucleotide of embodiments 1 to 20, wherein the contiguous nucleotide sequence includes or consists of a sequence selected from SEQ ID NO: 466, 640, 342, 287, and 566. 22. The oligonucleotide of embodiments 1 to 21, wherein the adjacent nucleotide sequence has zero to three mismatches compared to its complementary target nucleic acid. 23. The oligonucleotide of embodiment 22, wherein the adjacent nucleotide sequence has a mismatch compared to the target nucleic acid. 24. The oligonucleotide of embodiment 22, wherein the contiguous nucleotide sequence has two mismatches compared to the target nucleic acid. 25. The oligonucleotide of embodiment 22, wherein the adjacent nucleotide sequence is completely complementary to the target nucleic acid sequence. 26. The oligonucleotide of Examples 1 to 25, which includes one or more modified nucleosides. 27. The oligonucleotide of embodiment 26, wherein the one or more modified nucleosides are high-affinity modified nucleosides. 28. The oligonucleotide of embodiment 26 or 27, wherein the one or more modified nucleosides are 2'sugar modified nucleosides. 29. The oligonucleotide of embodiment 28, wherein the one or more 2'sugar-modified nucleosides are independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl Base-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, 2'-fluoro-ANA and LNA core Glycosides. 30. The oligonucleotide of embodiment 28, wherein the one or more modified nucleosides are LNA nucleosides. 31. The oligonucleotide of embodiment 30, wherein the modified LNA nucleoside is oxy-LNA. 32. The oligonucleotide of embodiment 31, wherein the modified nucleoside is β-D-oxy-LNA. 33. The oligonucleotide of embodiment 30, wherein the modified nucleoside is thio-LNA. 34. The oligonucleotide of embodiment 30, wherein the modified nucleoside is an amino-LNA. 35. The oligonucleotide of embodiment 30, wherein the modified nucleoside is cET. 36. The oligonucleotide of embodiment 30, wherein the modified nucleoside is ENA. 37. The oligonucleotide of embodiment 30, wherein the modified LNA nucleoside is selected from β-D-oxy-LNA, α-L-oxy-LNA, β-D-amino-LNA, α -L-amino-LNA, β-D-thio-LNA, α-L-thio-LNA, (S)cET, (R)cETβ-D-ENA and α-L-ENA. 38. The oligonucleotide of embodiments 30 to 37, wherein in addition to the modified LNA nucleoside, there is at least one 2'-substituted modified nucleoside. 39. The oligonucleotide of embodiment 38, wherein the 2'-substituted modified nucleoside is selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2 '-Alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-DNA, 2'-fluoro-ANA. 40. The oligonucleotide of any one of embodiments 1 to 39, wherein the oligonucleotide includes at least one modified internucleoside linkage. 41. The oligonucleotide of embodiment 40, wherein the modified internucleoside linkage is nuclease resistance. 42. The oligonucleotide of embodiment 40 or 41, wherein at least 50% of the internucleoside linkages in the adjacent nucleotide sequence are phosphorothioate internucleoside linkages or borane phosphate internucleoside linkages Link. 43. The oligonucleotide of embodiment 40 or 41, wherein all the internucleoside linkages in the adjacent nucleotide sequence are phosphorothioate internucleoside linkages. 44. The oligonucleotide of Examples 1 to 43, wherein the oligonucleotide can recruit RNase H. 45. The oligonucleotide of embodiment 44, wherein the oligonucleotide is a gapmer. 46. The oligonucleotide of embodiment 44 or 45, wherein the oligonucleotide is a gapmer of formula 5'-FG-F'-3', wherein regions F and F'independently include 1 to 7 Modified nucleosides or consist of them and G is a region capable of recruiting 6 to 16 nucleosides of RNaseH. 47. The oligonucleotide of embodiment 44 or 45, wherein the gapmer has the formula 5'-D'-FG-F'-3' or 5'-FG-F'-D''-3', Wherein regions F and F'independently include 1 to 7 modified nucleosides, G is a region capable of recruiting 6 to 16 nucleosides of RNaseH, and region D'or D'' includes 1 to 5 phosphodiester linkages. Nucleoside. 48. The oligonucleotide of embodiment 47, wherein D'or D'is optional. 49. The oligonucleotide of embodiment 47, wherein the region D'is composed of two nucleosides linked by phosphodiester. 50. The oligonucleotide of embodiment 49, wherein the nucleoside linked to the phosphodiester is ca (cytidine-adenosine). 51. The oligonucleotide of embodiment 46 or 47, wherein the modified nucleoside is a 2'sugar modified nucleoside independently selected from the group consisting of: 2'-O-alkyl-RNA, 2' -O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA ), 2'-fluoro-ANA and LNA nucleosides. 52. The oligonucleotide of embodiments 46 to 51, wherein one or more of the modified nucleosides in regions F and F'are LNA nucleosides. 53. The oligonucleotide of embodiment 52, wherein all the modified nucleosides in regions F and F'are LNA nucleosides. 54. The oligonucleotide of embodiment 53, wherein the regions F and F'are composed of LNA nucleosides. 55. The oligonucleotides of embodiments 52 to 54, wherein all the modified nucleosides in regions F and F'are oxy-LNA nucleosides. 56. The oligonucleotide of embodiment 52, wherein at least one of regions F or F'further comprises at least one 2'substituted modified nucleoside independently selected from the group consisting of: 2'-O-alkane -RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA and 2'-fluoro-DNA . 57. The oligonucleotides of embodiments 46 to 56, wherein the nucleosides recruiting RNaseH in region G are independently selected from DNA, α-L-LNA, C4'alkylated DNA, ANA and 2'F -ANA and UNA. 58. The oligonucleotide of embodiment 57, wherein the nucleosides in region G are DNA and/or α-L-LNA nucleosides. 59. The oligonucleotide of embodiment 57 or 58, wherein region G consists of at least 75% of DNA nucleosides. 60. The oligonucleotide of embodiments 1 to 59, wherein the oligonucleotide is selected from any one of CMP ID NO: 5_1 to 743_1 and 771_1 (Table 5). 61. The oligonucleotide of embodiments 1 to 60, wherein the oligonucleotide is selected from the group consisting of: CMP ID NO: 6_1, 8_1, 9_1, 13_1, 41_1, 42_1, 58_1, 77_1, 92_1, 111_1, 128_1, 151_1, 164_1, 166_1, 169_1, 171_1, 222_1, 233_1, 245_1, 246_1, 250_1, 251_1, 252_1, 256_1, 272_1, 273_1, 287_1, 292_1, 303_1, 314_1, 318_1, 320_1, 324_1, 336_1, 342_1, 343_1, 344_1, 345_1, 346_1, 349_1, 359_1, 360_1, 374_1, 408_1, 409_1, 415_1, 417_1, 424_1, 429_1, 430_1, 458_1, 464_1, 466_1, 474_1, 490_1, 493_1, 512_1, 519_1, 519_1, 529_1, 533_1, 534_1, 547_1, 566_1, 567_1, 578_1, 582_1, 601_1, 619_1, 620_1, 636_1, 637_1, 638_1, 640_1, 645_1, 650_1, 651_1, 652_1, 653_1, 658_1, 659_1, 660_1, 665_1, 678_1, 679_1, 680_1, 682_1, 683_1, 684_1, 687_1, 694_1, 706_1, 716_1, 728_1, 733_1, 734_1, and 735_1. 62. The oligonucleotide of embodiments 1 to 61, wherein the oligonucleotide is selected from the group consisting of: CMP ID NO: 287_1, 342_1, 466_1, 640_1, 566_1, 766_1, 767_1, 768_1, 769_1 and 770_1. 63. An antisense oligonucleotide conjugate comprising a. As the oligonucleotide of any one of Examples 1 to 62 (Region A); and b. At least one conjugate moiety (region C) that is covalently linked to the oligonucleotide. 64. The oligonucleotide conjugate of embodiment 63, wherein the conjugate part is selected from carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, Toxins, vitamins, viral proteins or combinations thereof. 65. The oligonucleotide conjugate of embodiment 63 or 64, wherein the conjugate portion contains a carbohydrate portion. 66. The oligonucleotide conjugate of embodiment 65, wherein the carbohydrate conjugate moiety comprises at least one targeted de-salivation covalently linked to the oligonucleotide of any one of embodiments 1 to 62 Part of the acid glycoprotein receptor. 67. The oligonucleotide conjugate of embodiment 66, wherein the portion of the conjugate targeted to the asialoglycoprotein receptor includes at least one carbohydrate portion selected from the group consisting of galactose, galactose Amine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-butyryl-galactosamine and N-isobutyrylgalactosamine. 68. The oligonucleotide conjugate of embodiment 66 or 67, wherein the part of the conjugate targeted to the asialoglycoprotein receptor is monovalent, divalent, trivalent or tetravalent. 69. The oligomer conjugate of embodiment 68, wherein the conjugate portion of the asialoglycoprotein receptor-targeting conjugate consists of two to four terminal GalNAc moieties, each GalNAc moiety is connected to a branching agent It is composed of molecular PEG spacer. 70. The oligonucleotide conjugate of embodiments 66 to 69, wherein the part of the conjugate targeted to the asialoglycoprotein receptor is a trivalent N-acetylgalactosamine (GalNAc) part. 71. The oligonucleotide conjugate of embodiments 66 to 70, wherein the conjugate part is selected from one of the trivalent GalNAc parts in FIG. 1. 72. The oligonucleotide conjugate of embodiment 71, wherein the conjugate part is the trivalent GalNAc part in FIG. 3. 73. The oligonucleotide conjugate of embodiments 63 to 72, wherein the linker is present between the oligonucleotide or adjacent oligonucleotide sequence and the conjugate part. 74. The oligonucleotide conjugate of embodiment 73, wherein the linking system is physiologically unstable linker (region B). 75. The oligonucleotide conjugate of embodiment 74, wherein the physiologically unstable ligation system nuclease susceptible linker. 76. The oligonucleotide conjugate of embodiment 74 or 75, wherein the physiologically unstable linkage system consists of 2 to 5 consecutive phosphodiester linkages. 77. The oligonucleotide conjugate of embodiment 76, wherein the physiologically unstable linker is equivalent to the region D'or D'shown in embodiments 47 to 50. 78. The oligonucleotide conjugate according to any one of embodiments 63 to 77, wherein the oligonucleotide conjugate is selected from CMP ID NO: 766_2, 767_2, 768_2, 769_2, and 770_2. 79. The oligonucleotide conjugate of embodiment 78, wherein the oligonucleotide conjugate is selected from the oligonucleotide conjugates shown in FIGS. 4, 5, 6, 7 and 8. 80. Oligonucleotide conjugates as in Examples 63 to 76, which show improved inhibition of PD-L1 in target cells compared with unconjugated oligonucleotides, or show improved inhibition between liver and spleen Cell distribution, or showing improved cellular uptake of conjugated oligonucleotides in the liver. 81. A pharmaceutical composition comprising the oligonucleotides of Examples 1 to 62 or the conjugates of Examples 63 to 80 and a pharmaceutically acceptable diluent, carrier, salt and/or adjuvant. 82. A method of manufacturing the oligonucleotides of Examples 1 to 62, which comprises reacting nucleotide units to thereby form adjacent nucleotides covalently linked included in the oligonucleotide unit. 83. The method of embodiment 82, which further comprises reacting the adjacent nucleotide sequence with the non-nucleotide conjugate moiety. 84. A method of manufacturing the composition of Example 81, which comprises mixing the oligonucleotide with a pharmaceutically acceptable diluent, carrier, salt, and/or adjuvant. 85. An in vivo or in vitro method for modulating the expression of PD-L1 in a target cell expressing PD-L1, the method comprising administering to the cell an effective amount of the oligonucleotides as in Examples 1 to 62 or as implemented The conjugate of Examples 63-80 or the pharmaceutical composition of Example 81. 86. A method for the treatment or prevention of a disease, which comprises administering to an individual suffering from or susceptible to the disease a therapeutically or preventively effective amount of oligonucleotides as in Examples 1 to 62 or as in Examples 63-80 Conjugate or pharmaceutical composition as in Example 81. 87. A method for restoring immunity against viruses or parasites, which comprises administering a therapeutically or preventively effective amount of oligonucleotide conjugates as in Examples 63 to 80 to an individual infected with the virus or parasite or as implemented The oligonucleotides of Examples 1 to 62 or the pharmaceutical composition of Example 81. 88. As in the method of Example 87, the restoration of immunity is to increase the CD8+ T cells specific for one or more HBV antigens in the liver when compared with the control. 89. The oligonucleotides of Examples 1 to 62 or the conjugates of Examples 63 to 80 or the pharmaceutical composition of Example 81 are used as agents for treating or preventing diseases in an individual. 90. The use of an oligonucleotide as in Examples 1 to 62 or a conjugate as in Examples 63 to 80 for the preparation of a medicament for the treatment or prevention of diseases in an individual. 91. The oligonucleotides of Examples 1 to 62 or the conjugates of Examples 63 to 80 or the pharmaceutical composition of Example 81 are used to restore immunity against viruses or parasites. 92. The use as in embodiment 91, wherein the restoration of immunity is to increase the CD8+ T cells specific to one or more HBV antigens in the liver when compared with the control. 93. The use as in embodiment 92, wherein the HBV antigen is HBsAg. 94. The method, oligonucleotide or use of embodiments 86 to 93, wherein the disease is related to the activity of PD-L1 in vivo. 95. The method, oligonucleotide or use of embodiments 86 to 94, wherein the disease is related to increased PD-L1 expression in antigen presenting cells. 96. The method, oligonucleotide or use of embodiment 95, wherein the oligonucleotides of embodiments 1 to 62 or the conjugates of embodiments 63 to 80 or the conjugates of embodiment 81 are used with untreated or in use Compared with the performance before the treatment of the pharmaceutical composition, the PD-L1 is reduced by at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%. 97. The method, oligonucleotide or use of embodiments 86 to 95, wherein the disease is selected from viral liver infection or parasitic infection. 98. The method, oligonucleotide or use of embodiment 98, wherein the viral infection is HBV, HCV or HDV. 99. The method, oligonucleotide or use of embodiments 86 to 95, wherein the disease is chronic HBV. 100. The method, oligonucleotide or use of embodiment 98, wherein the parasitic infection is malaria, toxoplasmosis, leishmaniasis or trypanosomiasis. 101. The method, oligonucleotide, or use of embodiments 86 to 100, wherein the system is mammalian. 102. The method, oligonucleotide or use of embodiment 101, wherein the mammal is a human. Examples Materials and methodsMotif Sequence and Oligonucleotide Compound Table 5: Oligonucleotide motif sequences (indicated by SEQ ID NO) targeting human PD-L1 transcript (SEQ ID NO: 1), the design of these sequences, and specific antisense oligos designed based on the motif sequence List of nucleotide compounds (indicated by CMP ID NO). SEQ ID NO Motif sequence design Oligonucleotide compounds CMP ID NO ID NO: 1 on the starting point dG 5 taattggctctactgc 2-11-3 TAattggctctacTGC 5_1 236 -20 6 tcgcataagaatgact 4-10-2 TCGCataagaatgaCT 6_1 371 -19 7 tgaacacacagtcgca 2-12-2 TGaacacacagtcgCA 7_1 382 -19 8 ctgaacacacagtcgc 3-10-3 CTGaacacacagtCGC 8_1 383 -twenty two 9 tctgaacacacagtcg 3-11-2 TCTgaacacacagtCG 9_1 384 -19 10 ttctgaacacacagtc 3-11-2 TTCtgaacacacagTC 10_1 385 -17 11 acaagtcatgttacta 2-11-3 ACaagtcatgttaCTA 11_1 463 -16 12 acacaagtcatgttac 2-12-2 ACacaagtcatgttAC 12_1 465 -14 13 cttacttagatgctgc 2-11-3 CTtacttagatgcTGC 13_1 495 -20 14 acttacttagatgctg 2-11-3 ACttacttagatgCTG 14_1 496 -18 15 gacttacttagatgct 3-11-2 GACttacttagatgCT 15_1 497 -19 16 agacttacttagatgc 2-11-3 AGacttacttagaTGC 16_1 498 -18 17 gcaggaagagacttac 3-10-3 GCAggaagagactTAC 17_1 506 -20 18 aataaattccgttcagg 4-9-4 AATAaattccgttCAGG 18_1 541 -twenty two 19 gcaaataaattccgtt 3-10-3 GCAaataaattccGTT 19_2 545 -18 19 gcaaataaattccgtt 4-8-4 GCAAataaattcCGTT 19_1 545 -20 20 agcaaataaattccgt 4-9-3 AGCAaataaattcCGT 20_1 546 -20 twenty one cagagcaaataaattcc 4-10-3 CAGAgcaaataaatTCC 21_1 548 -twenty one twenty two tggacagagcaaataaat 4-11-3 TGGAcagagcaaataAAT 22_1 551 -19 twenty three atggacagagcaaata 4-8-4 ATGGacagagcaAATA 23_1 554 -20 twenty four cagaatggacagagca 2-11-3 CAgaatggacagaGCA 24_1 558 -twenty one 25 ttctcagaatggacag 3-11-2 TTCtcagaatggacAG 25_1 562 -17 26 ctgaactttgacatag 4-8-4 CTGAactttgacATAG 26_1 663 -20 27 aagacaaacccagactga 2-13-3 AAgacaaacccagacTGA 27_1 675 -twenty one 28 tataagacaaacccagac 4-10-4 TATAagacaaacccAGAC 28_1 678 -twenty two 29 ttataagacaaacccaga 4-10-4 TTATaagacaaaccCAGA 29_1 679 -twenty three 30 tgttataagacaaaccc 4-10-3 TGTTataagacaaaCCC 30_1 682 -twenty two 31 tagaacaatggtacttt 4-9-4 TAGAacaatggtaCTTT 31_1 708 -20 32 gtagaacaatggtact 4-10-2 GTAGaacaatggtaCT 32_1 710 -19 33 aggtagaacaatggta 3-10-3 AGGtagaacaatgGTA 33_1 712 -19 34 aagaggtagaacaatgg 4-9-4 AAGAggtagaacaATGG 34_1 714 -twenty one 35 gcatccacagtaaatt 2-12-2 GCatccacagtaaaTT 35_1 749 -17 36 gaaggttatttaattc 2-11-3 GAaggttatttaaTTC 36_1 773 -13 37 ctaatcgaatgcagca 4-9-3 CTAAtcgaatgcaGCA 37_1 805 -twenty two 38 tacccaatctaatcga 3-10-3 TACccaatctaatCGA 38_1 813 -20 39 tagttacccaatctaa 3-10-3 TAGttacccaatcTAA 39_1 817 -19 40 catttagttacccaat 3-10-3 CATttagttacccAAT 40_1 821 -18 41 tcatttagttacccaa 3-10-3 TCAtttagttaccCAA 41_1 822 -19 42 ttcatttagttaccca 2-10-4 TTcatttagttaCCCA 42_1 823 -twenty two 43 gaattaatttcatttagt 4-10-4 GAATtaatttcattTAGT 43_1 829 -19 44 cagtgaggaattaattt 4-9-4 CAGTgaggaattaATTT 44_1 837 -20 45 ccaacagtgaggaatt 4-8-4 CCAAcagtgaggAATT 45_1 842 -twenty one 46 cccaacagtgaggaat 3-10-3 CCCaacagtgaggAAT 46_1 843 -twenty two 47 tatacccaacagtgagg 2-12-3 TAtacccaacagtgAGG 47_1 846 -twenty one 48 ttatacccaacagtgag 2-11-4 TTatacccaacagTGAG 48_1 847 -twenty one 49 tttatacccaacagtga 3-11-3 TTTatacccaacagTGA 49_1 848 -twenty one 50 cctttatacccaacag 3-10-3 CCTttatacccaaCAG 50_1 851 -twenty three 51 taacctttatacccaa 4-8-4 TAACctttatacCCAA 51_1 854 -twenty two 52 aataacctttataccca 3-10-4 AATaacctttataCCCA 52_1 855 -twenty three 53 gtaaataacctttata 3-11-2 GTAaataacctttaTA 53_1 859 -14 54 actgtaaataacctttat 4-10-4 ACTGtaaataacctTTAT 54_1 860 -20 55 atatatatgcaatgag 3-11-2 ATAtatatgcaatgAG 55_1 903 -14 56 agatatatatgcaatg 2-12-2 AGatatatatgcaaTG 56_1 905 -12 57 gagatatatatgcaat 3-10-3 GAGatatatatgcAAT 57_1 906 -15 58 ccagagatatatatgc 2-11-3 CCagagatatataTGC 58_1 909 -19 59 caatattccagagatat 4-9-4 CAATattccagagATAT 59_1 915 -20 60 gcaatattccagagata 4-10-3 GCAAtattccagagATA 60_1 916 -twenty two 61 agcaatattccagagat 3-11-3 AGCaatattccagaGAT 61_1 917 -twenty two 62 cagcaatattccagag 3-9-4 CAGcaatattccAGAG 62_1 919 -twenty two 63 aatcagcaatattccag 4-9-4 AATCagcaatattCCAG 63_1 921 -twenty three 64 acaatcagcaatattcc 4-9-4 ACAAtcagcaataTTCC 64_1 923 -twenty one 65 actaagtagttacacttct 2-14-3 ACtaagtagttacactTCT 65_1 957 -20 66 ctaagtagttacacttc 4-11-2 CTAAgtagttacactTC 66_1 958 -18 67 gactaagtagttacactt 3-12-3 GACtaagtagttacaCTT 67_1 959 -20 68 tgactaagtagttaca 3-9-4 TGActaagtagtTACA 68_1 962 -19 69 ctttgactaagtagtta 4-10-3 CTTTgactaagtagTTA 69_1 964 -19 70 ctctttgactaagtag 3-10-3 CTCtttgactaagTAG 70_1 967 -19 71 gctctttgactaagta 4-10-2 GCTCtttgactaagTA 71_1 968 -twenty one 72 ccttaaatactgttgac 2-11-4 CCttaaatactgtTGAC 72_1 1060 -20 73 cttaaatactgttgac 2-12-2 CTtaaatactgttgAC 73_1 1060 -13 74 tccttaaatactgttg 3-10-3 TCCttaaatactgTTG 74_1 1062 -18 75 tctccttaaatactgtt 4-11-2 TCTCcttaaatactgTT 75_1 1063 -19 76 tatcatagttctcctt 2-10-4 TAtcatagttctCCTT 76_1 1073 -twenty one 77 agtatcatagttctcc 3-10-3 AGTatcatagttcTCC 77_1 1075 -twenty two 78 gagtatcatagttctc 2-11-3 GAgtatcatagttCTC 78_1 1076 -18 79 agagtatcatagttct 2-10-4 AGagtatcatagTTCT 79_1 1077 -18 79 agagtatcatagttct 3-10-3 AGAgtatcatagtTCT 79_2 1077 -19 80 cagagtatcatagttc 3-10-3 CAGagtatcatagTTC 80_1 1078 -18 81 ttcagagtatcatagt 4-10-2 TTCAgagtatcataGT 81_1 1080 -18 82 cttcagagtatcatag 3-9-4 CTTcagagtatcATAG 82_1 1081 -19 83 ttcttcagagtatcata 4-11-2 TTCTtcagagtatcaTA 83_1 1082 -19 84 tttcttcagagtatcat 3-10-4 TTTcttcagagtaTCAT 84_1 1083 -20 85 gagaaaggctaagttt 4-9-3 GAGAaaggctaagTTT 85_1 1099 -19 86 gacactcttgtacatt 2-10-4 GAcactcttgtaCATT 86_1 1213 -19 87 tgagacactcttgtaca 2-13-2 TGagacactcttgtaCA 87_1 1215 -18 88 tgagacactcttgtac 2-11-3 TGagacactcttgTAC 88_1 1216 -18 89 ctttattaaactccat 2-10-4 CTttattaaactCCAT 89_1 1266 -18 90 accaaactttattaaa 4-10-2 ACCAaactttattaAA 90_1 1272 -14 91 aaacctctactaagtg 4-10-2 AAACctctactaagTG 91_1 1288 -16 92 agattaagacagttga 2-11-3 AGattaagacagtTGA 92_1 1310 -16 93 aagtaggagcaagaggc 2-12-3 AAgtaggagcaagaGGC 93_1 1475 -twenty two 94 aaagtaggagcaagagg 4-10-3 AAAGtaggagcaagAGG 94_1 1476 -20 95 gttaagcagccaggag 2-12-2 GTtaagcagccaggAG 95_1 1806 -20 96 agggtaggatgggtag 2-12-2 AGggtaggatgggtAG 96_1 1842 -20 97 aagggtaggatgggta 3-11-2 AAGggtaggatgggTA 97_1 1843 -20 98 caagggtaggatgggt 2-12-2 CAagggtaggatggGT 98_2 1844 -20 98 caagggtaggatgggt 3-11-2 CAAgggtaggatggGT 98_1 1844 -twenty one 99 ccaagggtaggatggg 2-12-2 CCaagggtaggatgGG 99_1 1845 -twenty two 100 tccaagggtaggatgg 2-12-2 TCcaagggtaggatGG 100_1 1846 -20 101 cttccaagggtaggat 4-10-2 CTTCcaagggtaggAT 101_1 1848 -twenty one 102 atcttccaagggtagga 3-12-2 ATCttccaagggtagGA 102_1 1849 -twenty two 103 agaagtgatggctcatt 2-11-4 AGaagtgatggctCATT 103_1 1936 -twenty one 104 aagaagtgatggctcat 3-10-4 AAGaagtgatggcTCAT 104_1 1937 -twenty one 105 gaagaagtgatggctca 3-11-3 GAAgaagtgatggcTCA 105_1 1938 -twenty one 106 atgaaatgtaaactggg 4-9-4 ATGAaatgtaaacTGGG 106_1 1955 -twenty one 107 caatgaaatgtaaactgg 4-10-4 CAATgaaatgtaaaCTGG 107_1 1956 -20 108 gcaatgaaatgtaaactg 4-10-4 GCAAtgaaatgtaaACTG 108_1 1957 -20 109 agcaatgaaatgtaaact 4-10-4 AGCAatgaaatgtaAACT 109_1 1958 -20 110 gagcaatgaaatgtaaac 4-10-4 GAGCaatgaaatgtAAAC 110_1 1959 -19 111 tgaattcccatatccga 2-12-3 TGaattcccatatcCGA 111_1 1992 -twenty two 112 agaattatgaccatat 2-11-3 AGaattatgaccaTAT 112_1 2010 -15 113 aggtaagaattatgacc 3-10-4 AGGtaagaattatGACC 113_1 2014 -twenty one 114 tcaggtaagaattatgac 4-10-4 TCAGgtaagaattaTGAC 114_1 2015 -twenty two 115 cttcaggtaagaattatg 4-10-4 CTTCaggtaagaatTATG 115_1 2017 -twenty one 116 tcttcaggtaagaatta 4-9-4 TCTTcaggtaagaATTA 116_1 2019 -20 117 cttcttcaggtaagaat 4-9-4 CTTCttcaggtaaGAAT 117_1 2021 -twenty one 118 tcttcttcaggtaagaa 4-10-3 TCTTcttcaggtaaGAA 118_1 2022 -20 119 tcttcttcaggtaaga 3-10-3 TCTtcttcaggtaAGA 119_1 2023 -20 120 tggtctaagagaagaag 3-10-4 TGGtctaagagaaGAAG 120_1 2046 -20 121 gttggtctaagagaag 4-9-3 GTTGgtctaagagAAG 121_1 2049 -19 123 cagttggtctaagagaa 2-11-4 CAgttggtctaagAGAA 123_1 2050 -20 124 gcagttggtctaagagaa 3-13-2 GCAgttggtctaagagAA 124_1 2050 -twenty two 122 agttggtctaagagaa 3-9-4 AGTtggtctaagAGAA 122_1 2050 -20 126 gcagttggtctaagaga 2-13-2 GCagttggtctaagaGA 126_1 2051 -twenty one 125 cagttggtctaagaga 4-10-2 CAGTtggtctaagaGA 125_1 2051 -twenty one 127 gcagttggtctaagag 2-11-3 GCagttggtctaaGAG 127_1 2052 -twenty one 128 ctcatatcagggcagt 2-10-4 CTcatatcagggCAGT 128_1 2063 -twenty four 129 cacacatgttctttaac 4-11-2 CACAcatgttctttaAC 129_1 2087 -18 130 taaatacacacatgttct 3-11-4 TAAatacacacatgTTCT 130_1 2092 -19 131 gtaaatacacacatgttc 4-11-3 GTAAatacacacatgTTC 131_1 2093 -19 132 tgtaaatacacacatgtt 4-10-4 TGTAaatacacacaTGTT 132_1 2094 -twenty two 133 gatcatgtaaatacacac 4-10-4 GATCatgtaaatacACAC 133_1 2099 -20 134 agatcatgtaaatacaca 4-10-4 AGATcatgtaaataCACA 134_1 2100 -twenty one 135 caaagatcatgtaaatacac 4-12-4 CAAAgatcatgtaaatACAC 135_1 2101 -19 136 acaaagatcatgtaaataca 4-12-4 ACAAagatcatgtaaaTACA 136_1 2102 -20 137 gaatacaaagatcatgta 4-10-4 GAATacaaagatcaTGTA 137_1 2108 -20 138 agaatacaaagatcatgt 4-10-4 AGAAtacaaagatcATGT 138_1 2109 -20 139 cagaatacaaagatcatg 4-10-4 CAGAatacaaagatCATG 139_1 2110 -twenty one 140 gcagaatacaaagatca 4-9-4 GCAGaatacaaagATCA 140_1 2112 -twenty two 141 aggcagaatacaaagat 4-11-2 AGGCagaatacaaagAT 141_1 2114 -19 142 aaggcagaatacaaaga 4-10-3 AAGGcagaatacaaAGA 142_1 2115 -19 143 attagtgagggacgaa 3-10-3 ATTagtgagggacGAA 143_1 2132 -18 144 cattagtgagggacga 2-11-3 CAttagtgagggaCGA 144_1 2133 -20 145 gagggtgatggattag 2-11-3 GAgggtgatggatTAG 145_1 2218 -19 146 ttaggagtaataaagg 2-10-4 TTaggagtaataAAGG 146_1 2241 -14 147 ttaatgaatttggttg 3-11-2 TTAatgaatttggtTG 147_1 2263 -13 148 ctttaatgaatttggt 2-12-2 CTttaatgaatttgGT 148_1 2265 -14 149 catggattacaactaa 4-10-2 CATGgattacaactAA 149_1 2322 -16 150 tcatggattacaacta 2-11-3 TCatggattacaaCTA 150_1 2323 -16 151 gtcatggattacaact 3-11-2 GTCatggattacaaCT 151_1 2324 -18 152 cattaaatctagtcat 2-10-4 CAttaaatctagTCAT 152_1 2335 -16 153 gacattaaatctagtca 4-10-3 GACAttaaatctagTCA 153_1 2336 -19 154 agggacattaaatcta 4-10-2 AGGGacattaaatcTA 154_1 2340 -18 155 caaagcattataacca 4-9-3 CAAAgcattataaCCA 155_1 2372 -18 156 acttactaggcagaag 2-10-4 ACttactaggcaGAAG 156_1 2415 -19 157 cagagttaactgtaca 4-10-2 CAGAgttaactgtaCA 157_1 2545 -20 158 ccagagttaactgtac 4-10-2 CCAGagttaactgtAC 158_1 2546 -20 159 gccagagttaactgta 2-12-2 GCcagagttaactgTA 159_1 2547 -20 160 tgggccagagttaact 2-12-2 TGggccagagttaaCT 160_1 2550 -twenty one 161 cagcatctatcagact 2-12-2 CAgcatctatcagaCT 161_1 2576 -19 162 tgaaataacatgagtcat 3-11-4 TGAaataacatgagTCAT 162_1 2711 -19 163 gtgaaataacatgagtc 3-10-4 GTGaaataacatgAGTC 163_1 2713 -19 164 tctgtttatgtcactg 4-10-2 TCTGtttatgtcacTG 164_1 2781 -20 165 gtctgtttatgtcact 4-10-2 GTCTgtttatgtcaCT 165_1 2782 -twenty two 166 tggtctgtttatgtca 2-10-4 TGgtctgtttatGTCA 166_1 2784 -twenty one 167 ttggtctgtttatgtc 4-10-2 TTGGtctgtttatgTC 167_1 2785 -20 168 tcacccattgtttaaa 2-12-2 TCacccattgtttaAA 168_1 2842 -15 169 ttcagcaaatattcgt 2-10-4 TTcagcaaatatTCGT 169_1 2995 -17 170 gtgtgttcagcaaatat 3-10-4 GTGtgttcagcaaATAT 170_1 2999 -twenty one 171 tctattgttaggtatc 3-10-3 TCTattgttaggtATC 171_1 3053 -18 172 attgcccatcttactg 2-12-2 ATtgcccatcttacTG 172_1 3118 -19 173 tattgcccatcttact 3-11-2 TATtgcccatcttaCT 173_1 3119 -twenty one 174 aaatattgcccatctt 2-11-3 AAatattgcccatCTT 174_1 3122 -17 175 ataaccttatcataca 3-11-2 ATAaccttatcataCA 175_1 3174 -16 176 tataaccttatcatac 2-11-3 TAtaaccttatcaTAC 176_1 3175 -14 177 ttataaccttatcata 3-11-2 TTAtaaccttatcaTA 177_1 3176 -14 178 tttataaccttatcat 3-10-3 TTTataaccttatCAT 178_1 3177 -16 179 actgctattgctatct 2-11-3 ACtgctattgctaTCT 179_1 3375 -19 180 aggactgctattgcta 2-11-3 AGgactgctattgCTA 180_1 3378 -twenty one 181 gaggactgctattgct 3-11-2 GAGgactgctattgCT 181_1 3379 -twenty two 182 acgtagaataataaca 2-12-2 ACgtagaataataaCA 182_1 3561 -11 183 ccaagtgatataatgg 2-10-4 CCaagtgatataATGG 183_1 3613 -19 184 ttagcagaccaagtga 2-10-4 TTagcagaccaaGTGA 184_1 3621 -twenty one 185 gtttagcagaccaagt 2-12-2 GTttagcagaccaaGT 185_1 3623 -19 186 tgacagtgattatatt 2-12-2 TGacagtgattataTT 186_1 3856 -13 187 tgtccaagatattgac 4-10-2 TGTCcaagatattgAC 187_1 3868 -18 188 gaatatcctagattgt 3-10-3 GAAtatcctagatTGT 188_1 4066 -18 189 caaactgagaatatcc 2-11-3 CAaactgagaataTCC 189_1 4074 -16 190 gcaaactgagaatatc 3-11-2 GCAaactgagaataTC 190_1 4075 -16 191 tcctattacaatcgta 3-11-2 TCCtattacaatcgTA 191_1 4214 -19 192 ttcctattacaatcgt 4-10-2 TTCCtattacaatcGT 192_1 4,215 -19 193 actaatgggaggattt 2-12-2 ACtaatgggaggatTT 193_1 4256 -15 194 tagttcagagaataag 2-12-2 TAgttcagagaataAG 194_1 4429 -13 195 taacatatagttcaga 2-11-3 TAacatatagttcAGA 195_1 4436 -15 196 ataacatatagttcag 3-11-2 ATAacatatagttcAG 196_1 4437 -14 197 cataacatatagttca 2-12-2 CAtaacatatagttCA 197_1 4438 -13 198 tcataacatatagttc 2-12-2 TCataacatatagtTC 198_1 4439 -12 199 tagctcctaacaatca 4-10-2 TAGCtcctaacaatCA 199_1 4507 -twenty two 200 ctccaatctttgtata 4-10-2 CTCCaatctttgtaTA 200_1 4602 -20 201 tctccaatctttgtat 4-10-2 TCTCcaatctttgtAT 201_1 4603 -19 202 tctatttcagccaatc 2-12-2 TCtatttcagccaaTC 202_1 4708 -17 203 cggaagtcagagtgaa 3-10-3 CGGaagtcagagtGAA 203_1 4782 -19 204 ttaagcatgaggaata 4-10-2 TTAAgcatgaggaaTA 204_1 4798 -16 205 tgattgagcacctctt 3-10-3 TGAttgagcacctCTT 205_1 4831 -twenty two 206 gactaattatttcgtt 3-11-2 GACtaattatttcgTT 206_1 4857 -15 207 tgactaattatttcgt 3-10-3 TGActaattatttCGT 207_1 4858 -17 208 gtgactaattatttcg 3-10-3 GTGactaattattTCG 208_1 4859 -17 209 ctgcttgaaatgtgac 4-10-2 CTGCttgaaatgtgAC 209_1 4870 -20 210 cctgcttgaaatgtga 2-11-3 CCtgcttgaaatgTGA 210_1 4871 -twenty one 211 atcctgcttgaaatgt 2-10-4 ATcctgcttgaaATGT 211_1 4873 -20 212 attataaatctattct 3-10-3 ATTataaatctatTCT 212_1 5027 -13 213 gctaaatactttcatc 2-11-3 GCtaaatactttcATC 213_1 5151 -16 214 cattgtaacataccta 2-10-4 CAttgtaacataCCTA 214_1 5251 -19 215 gcattgtaacatacct 2-12-2 GCattgtaacatacCT 215_1 5252 -18 216 taatattgcaccaaat 2-12-2 TAatattgcaccaaAT 216_1 5295 -13 217 gataatattgcaccaa 2-11-3 GAtaatattgcacCAA 217_1 5297 -16 218 agataatattgcacca 2-12-2 AGataatattgcacCA 218_1 5298 -16 219 gccaagaagataatat 2-10-4 GCcaagaagataATAT 219_1 5305 -17 220 cacagccacataaact 4-10-2 CACAgccacataaaCT 220_1 5406 -twenty one 221 ttgtaattgtggaaac 2-12-2 TTgtaattgtggaaAC 221_1 5463 -12 222 tgacttgtaattgtgg 2-11-3 TGacttgtaattgTGG 222_1 5467 -18 223 tctaactgaaatagtc 2-12-2 TCtaactgaaatagTC 223_1 5503 -13 224 gtggttctaactgaaa 3-11-2 GTGgttctaactgaAA 224_1 5508 -16 225 caatatgggacttggt 2-12-2 CAatatgggacttgGT 225_1 5522 -18 226 atgacaatatgggact 3-11-2 ATGacaatatgggaCT 226_1 5526 -17 227 tatgacaatatgggac 4-10-2 TATGacaatatgggAC 227_1 5527 -17 228 atatgacaatatggga 4-10-2 ATATgacaatatggGA 228_1 5528 -17 229 cttcacttaataatta 2-11-3 CTtcacttaataaTTA 229_1 5552 -13 230 ctgcttcacttaataa 4-10-2 CTGCttcacttaatAA 230_1 5555 -18 231 aagactgcttcactta 2-11-3 AAgactgcttcacTTA 231_1 5559 -17 232 gaatgccctaattatg 4-10-2 GAATgccctaattaTG 232_1 5589 -19 233 tggaatgccctaatta 3-11-2 TGGaatgccctaatTA 233_1 5591 -19 234 gcaaatgccagtaggt 3-11-2 GCAaatgccagtagGT 234_1 5642 -twenty three 235 ctaatggaaggatttg 3-11-2 CTAatggaaggattTG 235_1 5673 -15 236 aatatagaacctaatg 2-12-2 AAtatagaacctaaTG 236_1 5683 -10 237 gaaagaatagaatgtt 3-10-3 GAAagaatagaatGTT 237_1 5769 -12 238 atgggtaatagattat 3-11-2 ATGggtaatagattAT 238_1 5893 -15 239 gaaagagcacagggtg 2-12-2 GAaagagcacagggTG 239_1 6103 -18 240 ctacatagagggaatg 4-10-2 CTACatagagggaaTG 240_1 6202 -18 241 gcttcctacatagagg 2-10-4 GCttcctacataGAGG 241_1 6207 -twenty four 242 tgcttcctacatagag 4-10-2 TGCTtcctacatagAG 242_1 6208 -twenty two 243 tgggcttgaaatatgt 2-11-3 TGggcttgaaataTGT 243_1 6417 -19 244 cattatatttaagaac 3-11-2 CATtatatttaagaAC 244_1 6457 -11 245 tcggttatgttatcat 2-10-4 TCggttatgttaTCAT 245_1 6470 -19 246 cactttatctggtcgg 2-10-4 CActttatctggTCGG 246_1 6482 -twenty two 247 aaattggcacagcgtt 3-10-3 AAAttggcacagcGTT 247_1 6505 -18 248 accgtgacagtaaatg 4-9-3 ACCGtgacagtaaATG 248_1 6577 -20 249 tgggaaccgtgacagta 2-13-2 TGggaaccgtgacagTA 249_1 6581 -twenty two 250 ccacatataggtcctt 2-11-3 CCacatataggtcCTT 250_1 6597 -twenty one 251 catattgctaccatac 2-11-3 CAtattgctaccaTAC 251_1 6617 -18 252 tcatattgctaccata 3-10-3 TCAtattgctaccATA 252_1 6618 -19 253 caattgtcatattgct 4-8-4 CAATtgtcatatTGCT 253_1 6624 -twenty one 254 cattcaattgtcatattg 3-12-3 CATtcaattgtcataTTG 254_1 6626 -18 255 tttctactgggaatttg 4-9-4 TTTCtactgggaaTTTG 255_1 6644 -20 256 caattagtgcagccag 3-10-3 CAAttagtgcagcCAG 256_1 6672 -twenty one 257 gaataatgttcttatcc 4-10-3 GAATaatgttcttaTCC 257_1 6704 -20 258 cacaaattgaataatgttct 4-13-3 CACAaattgaataatgtTCT 258_1 6709 -20 259 catgcacaaattgaataat 4-11-4 CATGcacaaattgaaTAAT 259_1 6714 -20 260 atcctgcaatttcacat 3-11-3 ATCctgcaatttcaCAT 260_1 6832 -twenty two 261 ccaccatagctgatca 2-12-2 CCaccatagctgatCA 261_1 6868 -twenty two 262 accaccatagctgatca 2-12-3 ACcaccatagctgaTCA 262_1 6868 -twenty three 263 caccaccatagctgatc 2-13-2 CAccaccatagctgaTC 263_1 6869 -twenty one 264 tagtcggcaccaccat 2-12-2 TAgtcggcaccaccAT 264_1 6877 -twenty two 265 cttgtagtcggcaccac 1-14-2 CttgtagtcggcaccAC 265_1 6880 -twenty one 266 cttgtagtcggcacca 1-13-2 CttgtagtcggcacCA 266_1 6881 -twenty one 267 cgcttgtagtcggcac 2-12-2 CGcttgtagtcggcAC 267_1 6883 -twenty one 268 tcaataaagatcaggc 3-11-2 TCAataaagatcagGC 268_1 6942 -17 269 tggacttacaagaatg 2-12-2 TGgacttacaagaaTG 269_1 6986 -14 270 atggacttacaagaat 3-11-2 ATGgacttacaagaAT 270_1 6987 -15 271 gctcaagaaattggat 4-10-2 GCTCaagaaattggAT 271_1 7073 -19 272 tactgtagaacatggc 4-10-2 TACTgtagaacatgGC 272_1 7133 -twenty one 273 gcaattcatttgatct 4-9-3 GCAAttcatttgaTCT 273_1 7239 -20 274 tgaagggaggagggacac 2-14-2 TGaagggaggagggacAC 274_1 7259 -20 275 agtggtgaagggaggag 2-13-2 AGtggtgaagggaggAG 275_1 7265 -twenty one 276 tagtggtgaagggaggag 2-14-2 TAgtggtgaagggaggAG 276_1 7265 -twenty one 277 atagtggtgaagggaggag 1-16-2 AtagtggtgaagggaggAG 277_1 7265 -20 278 tagtggtgaagggagga 2-13-2 TAgtggtgaagggagGA 278_1 7266 -twenty one 279 atagtggtgaagggagga 2-14-2 ATagtggtgaagggagGA 279_1 7266 -twenty one 280 tagtggtgaagggagg 3-11-2 TAGtggtgaagggaGG 280_1 7267 -twenty one 281 atagtggtgaagggagg 3-12-2 ATAgtggtgaagggaGG 281_1 7267 -twenty two 282 gatagtggtgaagggagg 2-14-2 GAtagtggtgaagggaGG 282_1 7267 -twenty one 283 atagtggtgaagggag 4-10-2 ATAGtggtgaagggAG 283_1 7268 -20 284 gatagtggtgaagggag 2-12-3 GAtagtggtgaaggGAG 284_1 7268 -twenty one 285 gagatagtggtgaagg 2-10-4 GAgatagtggtgAAGG 285_1 7271 -20 286 catgggagatagtggt 4-10-2 CATGggagatagtgGT 286_1 7276 -twenty two 287 acaaataatggttactct 4-10-4 ACAAataatggttaCTCT 287_1 7302 -20 288 acacacaaataatggtta 4-10-4 ACACacaaataatgGTTA 288_1 7306 -20 289 gagggacacacaaataat 3-11-4 GAGggacacacaaaTAAT 289_1 7311 -twenty one 290 atatagagaggctcaa 4-8-4 ATATagagaggcTCAA 290_1 7390 -twenty one 291 ttgatatagagaggct 2-10-4 TTgatatagagaGGCT 291_1 7393 -20 292 gcatttgatatagaga 4-9-3 GCATttgatatagAGA 292_1 7397 -20 293 tttgcatttgatatag 2-11-3 TTtgcatttgataTAG 293_1 7400 -15 294 ctggaagaataggttc 3-11-2 CTGgaagaataggtTC 294_1 7512 -17 295 actggaagaataggtt 4-10-2 ACTGgaagaataggTT 295_1 7513 -18 296 tactggaagaataggt 4-10-2 TACTggaagaatagGT 296_1 7514 -18 297 tggcttatcctgtact 4-10-2 TGGCttatcctgtaCT 297_1 7526 -25 298 atggcttatcctgtac 2-10-4 ATggcttatcctGTAC 298_1 7527 -twenty two 299 tatggcttatcctgta 4-10-2 TATGgcttatcctgTA 299_1 7528 -twenty two 300 gtatggcttatcctgt 3-10-3 GTAtggcttatccTGT 300_1 7529 -twenty three 301 atgaatatatgcccagt 2-11-4 ATgaatatatgccCAGT 301_1 7547 -twenty two 302 gatgaatatatgccca 2-10-4 GAtgaatatatgCCCA 302_1 7549 -twenty two 303 caagatgaatatatgcc 3-10-4 CAAgatgaatataTGCC 303_1 7551 -twenty one 304 gacaacatcagtataga 4-9-4 GACAacatcagtaTAGA 304_1 7572 -twenty two 305 caagacaacatcagta 4-8-4 CAAGacaacatcAGTA 305_1 7576 -20 306 cactcctagttccttt 3-10-3 CACtcctagttccTTT 306_1 7601 -twenty two 307 aacactcctagttcct 3-10-3 AACactcctagttCCT 307_1 7603 -twenty two 308 taacactcctagttcc 2-11-3 TAacactcctagtTCC 308_1 7604 -20 309 ctaacactcctagttc 2-12-2 CTaacactcctagtTC 309_1 7605 -18 310 tgataacataactgtg 2-12-2 TGataacataactgTG 310_1 7637 -13 311 ctgataacataactgt 2-10-4 CTgataacataaCTGT 311_1 7638 -18 312 tttgaactcaagtgac 4-10-2 TTTGaactcaagtgAC 312_1 7654 -16 313 tcctttacttagctag 4-9-3 TCCTttacttagcTAG 313_1 7684 -twenty three 314 gagtttggattagctg 2-11-3 GAgtttggattagCTG 314_1 7764 -20 315 tgggatatgacaggga 2-11-3 TGggatatgacagGGA 315_1 7838 -twenty one 316 tgtgggatatgacagg 4-10-2 TGTGggatatgacaGG 316_1 7840 -twenty two 317 atatggaagggatatc 4-10-2 ATATggaagggataTC 317_1 7875 -17 318 acaggatatggaaggg 3-10-3 ACAggatatggaaGGG 318_1 7880 -twenty one 319 atttcaacaggatatgg 4-9-4 ATTTcaacaggatATGG 319_1 7885 -20 320 gagtaatttcaacagg 2-11-3 GAgtaatttcaacAGG 320_1 7891 -17 321 agggagtaatttcaaca 4-9-4 AGGGagtaatttcAACA 321_1 7893 -twenty two 322 attagggagtaatttca 4-9-4 ATTAgggagtaatTTCA 322_1 7896 -twenty one 323 cttactattagggagt 2-10-4 CTtactattaggGAGT 323_1 7903 -20 324 cagcttactattaggg 2-11-3 CAgcttactattaGGG 324_1 7906 -20 326 atttcagcttactattag 3-11-4 ATTtcagcttactaTTAG 326_1 7908 -20 325 tcagcttactattagg 3-10-3 TCAgcttactattAGG 325_1 7907 -20 327 ttcagcttactattag 2-10-4 TTcagcttactaTTAG 327_1 7908 -17 328 cagatttcagcttact 4-10-2 CAGAtttcagcttaCT 328_1 7913 -twenty one 329 gactacaactagaggg 3-11-2 GACtacaactagagGG 329_1 7930 -19 330 agactacaactagagg 4-10-2 AGACtacaactagaGG 330_1 7931 -19 331 aagactacaactagag 2-12-2 AAgactacaactagAG 331_1 7932 -13 332 atgatttaattctagtcaaa 4-12-4 ATGAtttaattctagtCAAA 332_1 7982 -20 333 tttaattctagtcaaa 3-10-3 TTTaattctagtcAAA 333_1 7982 -12 334 gatttaattctagtca 4-8-4 GATTtaattctaGTCA 334_1 7984 -20 771 tgatttaattctagtca 3-10-4 TGAtttaattctaGTCA 771_1 7984 -20 335 atgatttaattctagtca 4-11-3 ATGAtttaattctagTCA 335_1 7984 -20 336 gatgatttaattctagtca 4-13-2 GATGatttaattctagtCA 336_1 7984 -20 337 gatttaattctagtca 2-10-4 GAtttaattctaGTCA 337_1 7984 -18 338 gatgatttaattctagtc 4-11-3 GATGatttaattctaGTC 338_1 7985 -20 339 tgatttaattctagtc 2-12-2 TGatttaattctagTC 339_1 7985 -13 340 gagatgatttaattcta 4-9-4 GAGAtgatttaatTCTA 340_1 7988 -20 341 gagatgatttaattct 3-10-3 GAGatgatttaatTCT 341_1 7989 -16 342 cagattgatggtagtt 4-10-2 CAGAttgatggtagTT 342_1 8030 -19 343 ctcagattgatggtag 2-10-4 CTcagattgatgGTAG 343_1 8032 -20 344 gttagccctcagattg 3-10-3 GTTagccctcagaTTG 344_1 8039 -twenty three 345 tgtattgttagccctc 2-10-4 TGtattgttagcCCTC 345_1 8045 -twenty four 346 acttgtattgttagcc 2-10-4 ACttgtattgttAGCC 346_1 8048 -twenty two 347 agccagtatcagggac 3-11-2 AGCcagtatcagggAC 347_1 8191 -twenty three 348 ttgacaatagtggcat 2-10-4 TTgacaatagtgGCAT 348_1 8213 -20 349 acaagtggtatcttct 3-10-3 ACAagtggtatctTCT 349_1 8228 -19 350 aatctactttacaagt 4-10-2 AATCtactttacaaGT 350_1 8238 -16 351 cacagtagatgcctgata 2-12-4 CAcagtagatgcctGATA 351_1 8351 -twenty four 352 gaacacagtagatgcc 2-11-3 GAacacagtagatGCC 352_1 8356 -twenty one 353 cttggaacacagtagat 4-11-2 CTTGgaacacagtagAT 353_1 8359 -20 354 atatcttggaacacag 3-10-3 ATAtcttggaacaCAG 354_1 8364 -18 355 tctttaatatcttggaac 3-11-4 TCTttaatatcttgGAAC 355_1 8368 -19 356 tgatttctttaatatcttg 2-13-4 TGatttctttaatatCTTG 356_1 8372 -19 357 tgatgatttctttaatatc 2-13-4 TGatgatttctttaaTATC 357_1 8375 -18 358 aggctaagtcatgatg 3-11-2 AGGctaagtcatgaTG 358_1 8389 -19 359 ttgatgaggctaagtc 4-10-2 TTGAtgaggctaagTC 359_1 8395 -19 360 ccaggattatactctt 3-11-2 CCAggattatactcTT 360_1 8439 -20 361 gccaggattatactct 2-10-4 GCcaggattataCTCT 361_1 8440 -twenty three 362 ctgccaggattatact 3-11-2 CTGccaggattataCT 362_1 8442 -twenty one 363 cagaaacttatactttatg 4-13-2 CAGAaacttatactttaTG 363_1 8473 -19 364 aagcagaaacttatact 4-9-4 AAGCagaaacttaTACT 364_1 8478 -20 365 gaagcagaaacttatact 3-11-4 GAAgcagaaacttaTACT 365_1 8478 -20 366 tggaagcagaaacttatact 3-15-2 TGGaagcagaaacttataCT 366_1 8478 -twenty one 367 tggaagcagaaacttatac 3-13-3 TGGaagcagaaacttaTAC 367_1 8479 -20 368 aagcagaaacttatac 2-11-3 AAgcagaaacttaTAC 368_1 8479 -13 369 tggaagcagaaacttata 3-11-4 TGGaagcagaaactTATA 369_1 8480 -twenty one 370 aagggatattatggag 4-10-2 AAGGgatattatggAG 370_1 8587 -18 371 tgccggaagatttcct 2-12-2 TGccggaagatttcCT 371_1 8641 -twenty one 372 atggattgggagtaga 4-10-2 ATGGattgggagtaGA 372_1 8772 -twenty one 373 agatggattgggagta 2-12-2 AGatggattgggagTA 373_1 8774 -18 374 aagatggattgggagt 3-11-2 AAGatggattgggaGT 374_1 8775 -18 375 acaagatggattggga 2-10-4 ACaagatggattGGGA 375_1 8777 -20 375 acaagatggattggga 2-12-2 ACaagatggattggGA 375_2 8777 -17 376 agaaggttcagacttt 3-9-4 AGAaggttcagaCTTT 376_1 8835 -20 377 gcagaaggttcagact 2-11-3 GCagaaggttcagACT 377_1 8837 -twenty one 377 gcagaaggttcagact 3-11-2 GCAgaaggttcagaCT 377_2 8837 -twenty two 378 tgcagaaggttcagac 4-10-2 TGCAgaaggttcagAC 378_1 8838 -twenty two 379 agtgcagaaggttcag 2-11-3 AGtgcagaaggttCAG 379_1 8840 -20 379 agtgcagaaggttcag 4-10-2 AGTGcagaaggttcAG 379_2 8840 -twenty one 380 aagtgcagaaggttca 4-10-2 AAGTgcagaaggttCA 380_1 8841 -20 381 taagtgcagaaggttc 2-10-4 TAagtgcagaagGTTC 381_1 8842 -19 382 tctaagtgcagaaggt 2-10-4 TCtaagtgcagaAGGT 382_1 8844 -twenty one 383 ctcaggagttctacttc 3-12-2 CTCaggagttctactTC 383_1 8948 -20 384 ctcaggagttctactt 3-10-3 CTCaggagttctaCTT 384_1 8949 -twenty one 385 atggaggtgactcaggag 1-15-2 AtggaggtgactcaggAG 385_1 8957 -20 386 atggaggtgactcagga 2-13-2 ATggaggtgactcagGA 386_1 8958 -twenty one 387 atggaggtgactcagg 2-11-3 ATggaggtgactcAGG 387_1 8959 -twenty one 388 tatggaggtgactcagg 2-12-3 TAtggaggtgactcAGG 388_1 8959 -twenty one 389 atatggaggtgactcagg 2-14-2 ATatggaggtgactcaGG 389_1 8959 -twenty one 390 tatggaggtgactcag 4-10-2 TATGgaggtgactcAG 390_1 8960 -twenty one 391 atatggaggtgactcag 2-11-4 ATatggaggtgacTCAG 391_1 8960 -twenty two 392 catatggaggtgactcag 2-14-2 CAtatggaggtgactcAG 392_1 8960 -20 393 atatggaggtgactca 3-10-3 ATAtggaggtgacTCA 393_1 8961 -20 394 catatggaggtgactca 2-12-3 CAtatggaggtgacTCA 394_1 8961 -twenty one 395 catatggaggtgactc 2-10-4 CAtatggaggtgACTC 395_1 8962 -20 396 gcatatggaggtgactc 2-13-2 GCatatggaggtgacTC 396_1 8962 -twenty one 397 tgcatatggaggtgactc 2-14-2 TGcatatggaggtgacTC 397_1 8962 -twenty one 398 ttgcatatggaggtgactc 1-16-2 TtgcatatggaggtgacTC 398_1 8962 -20 399 tttgcatatggaggtgactc 1-17-2 TttgcatatggaggtgacTC 399_1 8962 -twenty one 400 gcatatggaggtgact 2-12-2 GCatatggaggtgaCT 400_1 8963 -20 401 tgcatatggaggtgact 2-13-2 TGcatatggaggtgaCT 401_1 8963 -20 402 ttgcatatggaggtgact 3-13-2 TTGcatatggaggtgaCT 402_1 8963 -twenty two 403 tttgcatatggaggtgact 1-16-2 TttgcatatggaggtgaCT 403_1 8963 -20 404 tgcatatggaggtgac 3-11-2 TGCatatggaggtgAC 404_1 8964 -20 405 ttgcatatggaggtgac 3-11-3 TTGcatatggaggtGAC 405_1 8964 -twenty one 406 tttgcatatggaggtgac 4-12-2 TTTGcatatggaggtgAC 406_1 8964 -twenty one 407 tttgcatatggaggtga 4-11-2 TTTGcatatggaggtGA 407_1 8965 -twenty one 408 tttgcatatggaggtg 2-10-4 TTtgcatatggaGGTG 408_1 8966 -twenty one 409 aagtgaagttcaacagc 2-11-4 AAgtgaagttcaaCAGC 409_1 8997 -20 410 tgggaagtgaagttca 2-10-4 TGggaagtgaagTTCA 410_1 9002 -20 411 atgggaagtgaagttc 2-11-3 ATgggaagtgaagTTC 411_1 9003 -17 412 gatgggaagtgaagtt 4-9-3 GATGggaagtgaaGTT 412_1 9004 -twenty one 413 ctgtgatgggaagtgaa 3-11-3 CTGtgatgggaagtGAA 413_1 9007 -20 414 attgagtgaatccaaa 3-10-3 ATTgagtgaatccAAA 414_1 9119 -14 415 aattgagtgaatccaa 2-10-4 AAttgagtgaatCCAA 415_1 9120 -16 416 gataattgagtgaatcc 4-10-3 GATAattgagtgaaTCC 416_1 9122 -20 417 gtgataattgagtgaa 3-10-3 GTGataattgagtGAA 417_1 9125 -16 418 aagaaaggtgcaataa 3-10-3 AAGaaaggtgcaaTAA 418_1 9155 -14 419 caagaaaggtgcaata 2-10-4 CAagaaaggtgcAATA 419_1 9156 -15 420 acaagaaaggtgcaat 4-10-2 ACAAgaaaggtgcaAT 420_1 9157 -16 421 atttaaactcacaaac 2-12-2 ATttaaactcacaaAC 421_1 9171 -10 422 ctgttaggttcagcga 2-10-4 CTgttaggttcaGCGA 422_1 9235 -twenty four 423 tctgaatgaacatttcg 4-9-4 TCTGaatgaacatTTCG 423_1 9260 -20 424 ctcattgaaggttctg 2-10-4 CTcattgaaggtTCTG 424_1 9281 -20 425 ctaatctcattgaagg 3-11-2 CTAatctcattgaaGG 425_1 9286 -17 426 cctaatctcattgaag 2-12-2 CCtaatctcattgaAG 426_1 9287 -16 427 actttgatctttcagc 3-10-3 ACTttgatctttcAGC 427_1 9305 -20 428 actatgcaacactttg 2-12-2 ACtatgcaacacttTG 428_1 9315 -15 429 caaatagctttatcgg 3-10-3 CAAatagctttatCGG 429_1 9335 -17 430 ccaaatagctttatcg 2-10-4 CCaaatagctttATCG 430_1 9336 -19 431 tccaaatagctttatc 4-10-2 TCCAaatagctttaTC 431_1 9337 -18 432 gatccaaatagcttta 4-10-2 GATCcaaatagcttTA 432_1 9339 -18 433 atgatccaaatagctt 2-10-4 ATgatccaaataGCTT 433_1 9341 -19 434 tatgatccaaatagct 4-10-2 TATGatccaaatagCT 434_1 9342 -18 435 taaacagggctgggaat 4-9-4 TAAAcagggctggGAAT 435_1 9408 -twenty two 436 acttaaacagggctgg 2-10-4 ACttaaacagggCTGG 436_1 9412 -twenty one 437 acacttaaacagggct 2-10-4 ACacttaaacagGGCT 437_1 9414 -twenty two 438 gaacacttaaacaggg 4-8-4 GAACacttaaacAGGG 438_1 9416 -20 439 agagaacacttaaacag 4-9-4 AGAGaacacttaaACAG 439_1 9418 -20 440 ctacagagaacactta 4-8-4 CTACagagaacaCTTA 440_1 9423 -20 441 atgctacagagaacact 3-10-4 ATGctacagagaaCACT 441_1 9425 -twenty two 442 ataaatgctacagagaaca 4-11-4 ATAAatgctacagagAACA 442_1 9427 -20 443 agataaatgctacagaga 2-12-4 AGataaatgctacaGAGA 443_1 9430 -20 444 tagagataaatgctaca 4-9-4 TAGAgataaatgcTACA 444_1 9434 -twenty one 445 tagatagagataaatgct 4-11-3 TAGAtagagataaatGCT 445_1 9437 -20 446 caatatactagatagaga 4-10-4 CAATatactagataGAGA 446_1 9445 -twenty one 447 tacacaatatactagatag 4-11-4 TACAcaatatactagATAG 447_1 9448 -twenty one 448 ctacacaatatactag 3-10-3 CTAcacaatatacTAG 448_1 9452 -16 449 gctacacaatatacta 4-8-4 GCTAcacaatatACTA 449_1 9453 -twenty one 450 atatgctacacaatatac 4-10-4 ATATgctacacaatATAC 450_1 9455 -20 451 tgatatgctacacaat 4-8-4 TGATatgctacaCAAT 451_1 9459 -20 452 atgatatgatatgctac 4-9-4 ATGAtatgatatgCTAC 452_1 9464 -twenty one 453 gaggagagagacaataaa 4-10-4 GAGGagagagacaaTAAA 453_1 9495 -20 454 ctaggaggagagagaca 3-11-3 CTAggaggagagagACA 454_1 9500 -twenty two 455 tattctaggaggagaga 4-10-3 TATTctaggaggagAGA 455_1 9504 -twenty one 456 ttatattctaggaggag 4-10-3 TTATattctaggagGAG 456_1 9507 -twenty one 457 gtttatattctaggag 3-9-4 GTTtatattctaGGAG 457_1 9510 -20 458 tggagtttatattctagg 2-12-4 TGgagtttatattcTAGG 458_1 9512 -twenty two 459 cgtaccaccactctgc 2-11-3 CGtaccaccactcTGC 459_1 9590 -25 460 tgaggaaatcattcattc 4-10-4 TGAGgaaatcattcATTC 460_1 9641 -twenty two 461 tttgaggaaatcattcat 4-10-4 TTTGaggaaatcatTCAT 461_1 9643 -20 462 aggctaatcctatttg 4-10-2 AGGCtaatcctattTG 462_1 9657 -twenty two 463 tttaggctaatcctat 4-8-4 TTTAggctaatcCTAT 463_1 9660 -twenty two 464 tgctccagtgtaccct 3-11-2 TGCtccagtgtaccCT 464_1 9755 -27 465 tagtagtactcgatag 2-10-4 TAgtagtactcgATAG 465_1 9813 -18 466 ctaattgtagtagtactc 3-12-3 CTAattgtagtagtaCTC 466_1 9818 -20 467 tgctaattgtagtagt 2-10-4 TGctaattgtagTAGT 467_1 9822 -19 468 agtgctaattgtagta 4-10-2 AGTGctaattgtagTA 468_1 9824 -19 469 gcaagtgctaattgta 4-10-2 GCAAgtgctaattgTA 469_1 9827 -20 470 gaggaaatgaactaattta 4-13-2 GAGGaaatgaactaattTA 470_1 9881 -18 471 caggaggaaatgaacta 4-11-2 CAGGaggaaatgaacTA 471_1 9886 -19 472 ccctagagtcatttcc 2-11-3 CCctagagtcattTCC 472_1 9902 -twenty four 473 atcttacatgatgaagc 3-11-3 ATCttacatgatgaAGC 473_1 9925 -20 475 agacacactcagatttcag 2-15-2 AGacacactcagatttcAG 475_1 9967 -20 474 gacacactcagatttcag 3-13-2 GACacactcagatttcAG 474_1 9967 -20 476 aagacacactcagatttcag 3-15-2 AAGacacactcagatttcAG 476_1 9967 -twenty one 477 agacacactcagatttca 2-13-3 AGacacactcagattTCA 477_1 9968 -20 478 aagacacactcagatttca 3-13-3 AAGacacactcagattTCA 478_1 9968 -twenty one 479 aaagacacactcagatttca 2-14-4 AAagacacactcagatTTCA 479_1 9968 -20 480 gaaagacacactcagatttc 3-14-3 GAAagacacactcagatTTC 480_1 9969 -20 481 aagacacactcagatttc 4-11-3 AAGAcacactcagatTTC 481_1 9969 -twenty one 482 aaagacacactcagatttc 4-11-4 AAAGacacactcagaTTTC 482_1 9969 -20 483 tgaaagacacactcagattt 4-14-2 TGAAagacacactcagatTT 483_1 9970 -20 484 tgaaagacacactcagatt 2-13-4 TGaaagacacactcaGATT 484_1 9971 -twenty one 485 tgaaagacacactcagat 3-12-3 TGAaagacacactcaGAT 485_1 9972 -20 486 attgaaagacacactca 4-10-3 ATTGaaagacacacTCA 486_1 9975 -19 487 tcattgaaagacacact 2-11-4 TCattgaaagacaCACT 487_1 9977 -18 488 ttccatcattgaaaga 3-9-4 TTCcatcattgaAAGA 488_1 9983 -18 489 ataataccacttatcat 4-9-4 ATAAtaccacttaTCAT 489_1 10010 -20 490 ttacttaatttctttgga 2-12-4 TTacttaatttcttTGGA 490_1 10055 -20 491 ttagaactagctttatca 3-12-3 TTAgaactagctttaTCA 491_1 10101 -20 492 gaggtacaaatatagg 3-10-3 GAGgtacaaatatAGG 492_1 10171 -18 493 cttatgatacaactta 3-10-3 CTTatgatacaacTTA 493_1 10384 -15 494 tcttatgatacaactt 2-11-3 TCttatgatacaaCTT 494_1 10385 -15 495 ttcttatgatacaact 3-11-2 TTCttatgatacaaCT 495_1 10386 -15 496 cagtttcttatgatac 2-11-3 CAgtttcttatgaTAC 496_1 10390 -16 497 gcagtttcttatgata 3-11-2 GCAgtttcttatgaTA 497_1 10391 -19 498 tacaaatgtctattaggtt 4-12-3 TACAaatgtctattagGTT 498_1 10457 -twenty one 499 tgtacaaatgtctattag 4-11-3 TGTAcaaatgtctatTAG 499_1 10460 -20 500 agcatcacaattagta 3-11-2 AGCatcacaattagTA 500_1 10535 -18 501 ctaatgatagtgaagc 3-11-2 CTAatgatagtgaaGC 501_1 10548 -17 502 agctaatgatagtgaa 3-11-2 AGCtaatgatagtgAA 502_1 10550 -16 503 atgccttgacatatta 4-10-2 ATGCcttgacatatTA 503_1 10565 -20 504 ctcaagattattgacac 4-9-4 CTCAagattattgACAC 504_1 10623 -20 505 acctcaagattattga 2-10-4 ACctcaagattaTTGA 505_2 10626 -18 505 acctcaagattattga 3-9-4 ACCtcaagattaTTGA 505_1 10626 -20 506 aacctcaagattattg 4-10-2 AACCtcaagattatTG 506_1 10627 -17 507 cacaaacctcaagattatt 4-13-2 CACAaacctcaagattaTT 507_1 10628 -20 508 gtacttaattagacct 3-9-4 GTActtaattagACCT 508_1 10667 -twenty one 509 agtacttaattagacc 4-9-3 AGTActtaattagACC 509_1 10668 -20 510 gtatgaggtggtaaac 4-10-2 GTATgaggtggtaaAC 510_1 10688 -18 511 aggaaacagcagaagtg 2-11-4 AGgaaacagcagaAGTG 511_1 10723 -twenty one 512 gcacaacccagaggaa 2-12-2 GCacaacccagaggAA 512_1 10735 -20 513 caagcacaacccagag 3-11-2 CAAgcacaacccagAG 513_1 10738 -20 514 ttcaagcacaacccag 3-10-3 TTCaagcacaaccCAG 514_1 10740 -twenty one 515 aattcaagcacaaccc 2-10-4 AAttcaagcacaACCC 515_1 10742 -20 516 taataattcaagcacaacc 4-13-2 TAATaattcaagcacaaCC 516_1 10743 -20 517 actaataattcaagcac 4-9-4 ACTAataattcaaGCAC 517_1 10747 -20 518 ataatactaataattcaagc 4-12-4 ATAAtactaataattcAAGC 518_1 10749 -19 519 tagatttgtgaggtaa 2-10-4 TAgatttgtgagGTAA 519_1 11055 -18 520 agccttaattctccat 4-10-2 AGCCttaattctccAT 520_1 11091 -twenty four 521 aatgatctagagcctta 4-9-4 AATGatctagagcCTTA 521_1 11100 -twenty two 522 ctaatgatctagagcc 3-10-3 CTAatgatctagaGCC 522_1 11103 -twenty two 523 actaatgatctagagc 3-9-4 ACTaatgatctaGAGC 523_1 11104 -twenty one 524 cattaacatgttcttatt 3-11-4 CATtaacatgttctTATT 524_1 11165 -19 525 acaagtacattaacatgttc 4-12-4 ACAAgtacattaacatGTTC 525_1 11170 -twenty two 526 ttacaagtacattaacatg 4-11-4 TTACaagtacattaaCATG 526_1 11173 -20 527 gctttattcatgtttat 4-9-4 GCTTtattcatgtTTAT 527_1 11195 -twenty two 528 gctttattcatgttta 3-11-2 GCTttattcatgttTA 528_1 11196 -18 529 agagctttattcatgttt 3-13-2 AGAgctttattcatgtTT 529_1 11197 -20 530 ataagagctttattcatg 4-10-4 ATAAgagctttattCATG 530_1 11200 -twenty one 531 cataagagctttattca 4-9-4 CATAagagctttaTTCA 531_1 11202 -twenty one 532 agcataagagctttat 4-8-4 AGCAtaagagctTTAT 532_1 11205 -twenty two 533 tagattgtttagtgca 3-10-3 TAGattgtttagtGCA 533_1 11228 -20 534 gtagattgtttagtgc 2-10-4 GTagattgtttaGTGC 534_1 11229 -twenty one 535 gacaattctagtagatt 4-9-4 GACAattctagtaGATT 535_1 11238 -twenty one 536 ctgacaattctagtag 3-9-4 CTGacaattctaGTAG 536_1 11241 -20 537 gctgacaattctagta 4-10-2 GCTGacaattctagTA 537_1 11242 -twenty one 538 aggattaagatacgta 2-12-2 AGgattaagatacgTA 538_1 11262 -15 539 caggattaagatacgt 2-11-3 CAggattaagataCGT 539_1 11263 -17 540 tcaggattaagatacg 3-11-2 TCAggattaagataCG 540_1 11264 -16 541 ttcaggattaagatac 2-10-4 TTcaggattaagATAC 541_1 11265 -15 542 aggaagaaagtttgattc 4-10-4 AGGAagaaagtttgATTC 542_1 11308 -twenty one 543 tcaaggaagaaagtttga 4-10-4 TCAAggaagaaagtTTGA 543_1 11311 -20 544 ctcaaggaagaaagtttg 4-10-4 CTCAaggaagaaagTTTG 544_1 11312 -20 545 tgctcaaggaagaaagt 3-10-4 TGCtcaaggaagaAAGT 545_1 11315 -twenty one 546 aattatgctcaaggaaga 4-11-3 AATTatgctcaaggaAGA 546_1 11319 -20 547 taggataccacattatga 4-12-2 TAGGataccacattatGA 547_1 11389 -twenty two 548 cataatttattccattcctc 2-15-3 CAtaatttattccattcCTC 548_1 11449 -twenty two 549 tgcataatttattccat 4-10-3 TGCAtaatttattcCAT 549_1 11454 -twenty two 550 actgcataatttattcc 4-10-3 ACTGcataatttatTCC 550_1 11456 -twenty one 551 ctaaactgcataatttatt 4-11-4 CTAAactgcataattTATT 551_1 11458 -20 552 ataactaaactgcata 2-10-4 ATaactaaactgCATA 552_1 11465 -16 553 ttattaataactaaactgc 3-12-4 TTAttaataactaaaCTGC 553_1 11468 -19 554 tagtacattattaataact 4-13-2 TAGTacattattaataaCT 554_1 11475 -18 555 cataactaaggacgtt 4-10-2 CATAactaaggacgTT 555_1 11493 -17 556 tcataactaaggacgt 2-11-3 TCataactaaggaCGT 556_1 11494 -16 557 cgtcataactaaggac 4-10-2 CGTCataactaaggAC 557_1 11496 -17 558 tcgtcataactaagga 2-12-2 TCgtcataactaagGA 558_1 11497 -16 559 atcgtcataactaagg 2-10-4 ATcgtcataactAAGG 559_1 11498 -17 560 gttagtatcttacatt 2-11-3 GTtagtatcttacATT 560_1 11525 -15 561 ctctattgttagtatc 3-10-3 CTCtattgttagtATC 561_1 11532 -17 562 agtatagagttactgt 3-10-3 AGTatagagttacTGT 562_1 11567 -19 563 ttcctggtgatacttt 4-10-2 TTCCtggtgatactTT 563_1 11644 -twenty one 564 gttcctggtgatactt 4-10-2 GTTCctggtgatacTT 564_1 11645 -twenty one 565 tgttcctggtgatact 2-12-2 TGttcctggtgataCT 565_1 11646 -20 566 ataaacatgaatctctcc 2-12-4 ATaaacatgaatctCTCC 566_1 11801 -20 567 ctttataaacatgaatctc 3-12-4 CTTtataaacatgaaTCTC 567_1 11804 -19 568 ctgtctttataaacatg 3-10-4 CTGtctttataaaCATG 568_1 11810 -19 569 ttgttataaatctgtctt 2-12-4 TTgttataaatctgTCTT 569_1 11820 -18 570 ttaaatttattcttggata 3-12-4 TTAaatttattcttgGATA 570_1 11849 -19 571 cttaaatttattcttgga 2-12-4 CTtaaatttattctTGGA 571_1 11851 -19 572 cttcttaaatttattcttg 4-13-2 CTTCttaaatttattctTG 572_1 11853 -18 573 tatgtttctcagtaaag 4-9-4 TATGtttctcagtAAAG 573_1 11877 -19 574 gaattatctttaaacca 3-10-4 GAAttatctttaaACCA 574_1 11947 -18 575 cccttaaatttctaca 3-11-2 CCCttaaatttctaCA 575_1 11980 -20 576 acactgctcttgtacc 4-10-2 ACACtgctcttgtaCC 576_1 11995 -twenty three 577 tgacaacactgctctt 3-10-3 TGAcaacactgctCTT 577_1 12000 -twenty one 578 tacatttattgggctc 4-10-2 TACAtttattgggcTC 578_1 12081 -19 579 gtacatttattgggct 2-10-4 GTacatttattgGGCT 579_1 12082 -twenty three 580 ttggtacatttattgg 3-10-3 TTGgtacatttatTGG 580_1 12085 -18 581 catgttggtacatttat 4-10-3 CATGttggtacattTAT 581_1 12088 -twenty one 582 aatcatgttggtacat 4-10-2 AATCatgttggtacAT 582_1 12092 -16 583 aaatcatgttggtaca 2-12-2 AAatcatgttggtaCA 583_1 12093 -14 584 gacaagtttggattaa 3-11-2 GACaagtttggattAA 584_1 12132 -14 585 aatgttcagatgcctc 2-10-4 AAtgttcagatgCCTC 585_1 12197 -twenty one 586 gcttaatgttcagatg 2-12-2 GCttaatgttcagaTG 586_1 12201 -17 587 cgtacatagcttgatg 4-10-2 CGTAcatagcttgaTG 587_1 12267 -20 588 gtgaggaattaggata 3-11-2 GTGaggaattaggaTA 588_1 12753 -17 589 gtaacaatatggtttg 3-11-2 GTAacaatatggttTG 589_1 12780 -15 590 gaaatattgtagacta 2-11-3 GAaatattgtagaCTA 590_1 13151 -14 591 ttgaaatattgtagac 3-11-2 TTGaaatattgtagAC 591_1 13153 -12 592 aagtctagtaatttgc 2-10-4 AAgtctagtaatTTGC 592_1 13217 -17 593 gctcagtagattataa 4-10-2 GCTCagtagattatAA 593_1 13259 -17 594 catacactgttgctaa 3-10-3 CATacactgttgcTAA 594_1 13296 -19 595 atggtctcaaatcatt 3-10-3 ATGgtctcaaatcATT 595_1 13314 -17 596 caatggtctcaaatca 4-10-2 CAATggtctcaaatCA 596_1 13316 -18 597 ttcctattgattgact 4-10-2 TTCCtattgattgaCT 597_1 13568 -20 598 tttctgttcacaacac 4-10-2 TTTCtgttcacaacAC 598_1 13,600 -17 599 aggaacccactaatct 2-11-3 AGgaacccactaaTCT 599_1 13702 -20 600 taaatggcaggaaccc 3-11-2 TAAatggcaggaacCC 600_1 13710 -19 601 gtaaatggcaggaacc 4-10-2 GTAAatggcaggaaCC 601_1 13711 -20 602 ttgtaaatggcaggaa 2-11-3 TTgtaaatggcagGAA 602_1 13713 -16 603 ttatgagttaggcatg 2-10-4 TTatgagttaggCATG 603_1 13835 -19 604 ccaggtgaaactttaa 3-11-2 CCAggtgaaactttAA 604_1 13935 -17 605 cccttagtcagctcct 3-10-3 CCCttagtcagctCCT 605_1 13,997 -30 606 acccttagtcagctcc 2-10-4 ACccttagtcagCTCC 606_1 13998 -27 607 cacccttagtcagctc 2-11-3 CAcccttagtcagCTC 607_1 13,999 -twenty four 608 tctcttactaggctcc 3-10-3 TCTcttactaggcTCC 608_1 14091 -twenty four 609 cctatctgtcatcatg 2-11-3 CCtatctgtcatcATG 609_1 14178 -20 610 tcctatctgtcatcat 3-11-2 TCCtatctgtcatcAT 610_1 14179 -20 611 gagaagtgtgagaagc 3-11-2 GAGaagtgtgagaaGC 611_1 14,808 -19 612 catccttgaagtttag 4-10-2 CATCcttgaagtttAG 612_1 14,908 -19 613 taataagatggctccc 3-10-3 TAAtaagatggctCCC 613_1 15046 -twenty one 614 caaggcataataagat 3-11-2 CAAggcataataagAT 614_1 15053 -14 615 ccaaggcataataaga 2-10-4 CCaaggcataatAAGA 615_1 15054 -18 616 tgatccaattctcacc 2-12-2 TGatccaattctcaCC 616_1 15151 -19 617 atgatccaattctcac 3-10-3 ATGatccaattctCAC 617_1 15152 -19 618 cgcttcatcttcaccc 3-11-2 CGCttcatcttcacCC 618_1 15260 -26 619 tatgacactgcatctt 2-10-4 TAtgacactgcaTCTT 619_1 15317 -19 620 gtatgacactgcatct 3-10-3 GTAtgacactgcaTCT 620_1 15318 -twenty one 621 tgtatgacactgcatc 2-10-4 TGtatgacactgCATC 621_1 15319 -20 622 ttctcttctgtaagtc 4-10-2 TTCTcttctgtaagTC 622_1 15363 -19 623 ttctacagaggaacta 2-10-4 TTctacagaggaACTA 623_1 15467 -17 624 actacagttctacaga 3-10-3 ACTacagttctacAGA 624_1 15,474 -19 625 ttcccacaggtaaatg 4-10-2 TTCCcacaggtaaaTG 625_1 15561 -twenty one 626 attatttgaatatactcatt 4-12-4 ATTAtttgaatatactCATT 626_1 15594 -20 627 tgggaggaaattatttg 4-10-3 TGGGaggaaattatTTG 627_1 15606 -20 628 tgactcatcttaaatg 4-10-2 TGACtcatcttaaaTG 628_1 15621 -17 629 ctgactcatcttaaat 3-11-2 CTGactcatcttaaAT 629_1 15622 -16 630 tttactctgactcatc 3-10-3 TTTactctgactcATC 630_1 15628 -17 631 tattggaggaattatt 3-11-2 TATtggaggaattaTT 631_1 15642 -14 632 gtattggaggaattat 3-11-2 GTAttggaggaattAT 632_1 15643 -16 633 tggtatacttctctaagtat 2-15-3 TGgtatacttctctaagTAT 633_1 15655 -twenty two 634 gatctcttggtatact 4-10-2 GATCtcttggtataCT 634_1 15666 -20 635 cagacaactctatacc 2-12-2 CAgacaactctataCC 635_1 15689 -18 636 aacatcagacaactcta 4-9-4 AACAtcagacaacTCTA 636_1 15693 -twenty one 637 taacatcagacaactc 4-10-2 TAACatcagacaacTC 637_1 15695 -16 638 tttaacatcagacaactc 4-10-4 TTTAacatcagacaACTC 638_1 15695 -20 639 atttaacatcagacaa 2-12-2 ATttaacatcagacAA 639_1 15698 -11 640 cctatttaacatcagac 2-11-4 CCtatttaacatcAGAC 640_1 15,700 -20 641 tccctatttaacatca 3-10-3 TCCctatttaacaTCA 641_1 15703 -twenty one 642 tcaacgactattggaat 4-9-4 TCAAcgactattgGAAT 642_1 15737 -20 643 cttatattctggctat 4-9-3 CTTAtattctggcTAT 643_1 15,850 -20 644 atccttatattctggc 4-10-2 ATCCttatattctgGC 644_1 15853 -twenty three 645 gatccttatattctgg 2-10-4 GAtccttatattCTGG 645_1 15854 -twenty one 646 tgatccttatattctg 3-10-3 TGAtccttatattCTG 646_1 15,855 -19 647 attgaaacttgatcct 4-8-4 ATTGaaacttgaTCCT 647_1 15864 -twenty one 648 actgtcattgaaactt 2-10-4 ACtgtcattgaaACTT 648_1 15870 -16 649 tcttactgtcattgaa 3-11-2 TCTtactgtcattgAA 649_1 15874 -16 650 aggatcttactgtcatt 2-11-4 AGgatcttactgtCATT 650_1 15877 -twenty one 651 gcaaatcaactccatc 3-10-3 GCAaatcaactccATC 651_1 15896 -20 652 gtgcaaatcaactcca 3-10-3 GTGcaaatcaactCCA 652_1 15898 -twenty two 653 caattatttctttgtgc 4-10-3 CAATtatttctttgTGC 653_1 15910 -twenty one 654 tggcaacaattatttctt 3-11-4 TGGcaacaattattTCTT 654_1 15915 -twenty one 655 gctggcaacaattatt 3-9-4 GCTggcaacaatTATT 655_1 15919 -twenty one 656 atccatttctactgcc 4-10-2 ATCCatttctactgCC 656_1 15973 -twenty four 657 taatatctattgatttcta 4-11-4 TAATatctattgattTCTA 657_1 15988 -20 658 tcaatagtgtagggca 2-12-2 TCaatagtgtagggCA 658_1 16093 -18 659 ttcaatagtgtagggc 3-11-2 TTCaatagtgtaggGC 659_1 16094 -19 660 aggttaattaattcaatag 4-11-4 AGGTtaattaattcaATAG 660_1 16102 -twenty one 661 catttgtaatccctag 3-10-3 CATttgtaatcccTAG 661_2 16163 -20 661 catttgtaatccctag 3-9-4 CATttgtaatccCTAG 661_1 16163 -twenty two 662 acatttgtaatcccta 3-10-3 ACAtttgtaatccCTA 662_1 16164 -20 663 aacatttgtaatccct 2-10-4 AAcatttgtaatCCCT 663_2 16165 -twenty one 663 aacatttgtaatccct 3-9-4 AACatttgtaatCCCT 663_1 16165 -twenty two 664 taaatttcaagttctg 2-11-3 TAaatttcaagttCTG 664_1 16184 -14 665 gtttaaatttcaagttct 3-11-4 GTTtaaatttcaagTTCT 665_1 16185 -19 666 ccaagtttaaatttcaag 4-10-4 CCAAgtttaaatttCAAG 666_1 16189 -twenty one 667 acccaagtttaaatttc 4-9-4 ACCCaagtttaaaTTTC 667_1 16192 -twenty two 668 catacagtgacccaagttt 2-14-3 CAtacagtgacccaagTTT 668_1 16199 -twenty three 669 acatcccatacagtga 2-11-3 ACatcccatacagTGA 669_1 16208 -twenty one 670 agcacagctctacatc 2-10-4 AGcacagctctaCATC 670_1 16219 -twenty two 671 atatagcacagctcta 3-9-4 ATAtagcacagcTCTA 671_1 16223 -twenty one 672 tccatatagcacagct 3-11-2 TCCatatagcacagCT 672_1 16226 -twenty two 673 atttccatatagcaca 3-9-4 ATTtccatatagCACA 673_1 16229 -20 674 tttatttccatatagca 4-9-4 TTTAtttccatatAGCA 674_1 16231 -twenty two 675 tttatttccatatagc 3-10-3 TTTatttccatatAGC 675_1 16232 -18 676 aaggagaggagattatg 4-9-4 AAGGagaggagatTATG 676_1 16409 -twenty one 677 agttcttgtgttagct 3-11-2 AGTtcttgtgttagCT 677_1 16456 -twenty one 678 gagttcttgtgttagc 2-12-2 GAgttcttgtgttaGC 678_1 16457 -20 679 attaattatccatccac 3-10-4 ATTaattatccatCCAC 679_1 16590 -twenty one 680 atcaattaattatccatc 3-11-4 ATCaattaattatcCATC 680_1 16593 -19 681 agaatcaattaattatcc 3-12-3 AGAatcaattaattaTCC 681_1 16596 -18 682 tgagataccgtgcatg 2-12-2 TGagataccgtgcaTG 682_1 16656 -18 683 aatgagataccgtgca 2-10-4 AAtgagataccgTGCA 683_1 16658 -twenty one 684 ctgtggttaggctaat 3-11-2 CTGtggttaggctaAT 684_1 16,834 -19 685 aagagtaagggtctgtggtt 1-17-2 AagagtaagggtctgtggTT 685_1 16842 -twenty one 686 gatgggttaagagtaa 4-9-3 GATGggttaagagTAA 686_1 16854 -19 687 agcagatgggttaaga 3-11-2 AGCagatgggttaaGA 687_1 16858 -20 688 tgtaaacatttgtagc 2-10-4 TGtaaacatttgTAGC 688_1 16886 -19 689 cctgcttataaatgta 3-11-2 CCTgcttataaatgTA 689_1 16898 -19 690 tgccctgcttataaat 4-10-2 TGCCctgcttataaAT 690_1 16901 -twenty three 691 tcttcttagttcaata 2-12-2 TCttcttagttcaaTA 691_1 16935 -15 692 tggtttctaactacat 2-10-4 TGgtttctaactACAT 692_1 16,980 -18 693 agtttggtttctaacta 2-12-3 AGtttggtttctaaCTA 693_1 16,983 -19 694 gaatgaaacttgcctg 3-10-3 GAAtgaaacttgcCTG 694_1 17047 -18 695 attatccttacatgat 3-10-3 ATTatccttacatGAT 695_1 17173 -17 696 gtacccaattatcctt 2-11-3 GTacccaattatcCTT 696_1 17,180 -twenty one 697 tgtacccaattatcct 3-10-3 TGTacccaattatCCT 697_1 17,181 -twenty four 698 ttgtacccaattatcc 2-11-3 TTgtacccaattaTCC 698_1 17,182 -20 699 tttgtacccaattatc 3-11-2 TTTgtacccaattaTC 699_1 17183 -17 700 agcagcaggttatatt 4-10-2 AGCAgcaggttataTT 700_1 17,197 -twenty two 701 tgggaagtggtctggg 3-10-3 TGGgaagtggtctGGG 701_1 17,292 -25 702 ctggagagtgataata 3-11-2 CTGgagagtgataaTA 702_1 17322 -17 703 aatgctggattacgtc 4-10-2 AATGctggattacgTC 703_1 17,354 -19 704 caatgctggattacgt 2-11-3 CAatgctggattaCGT 704_1 17355 -19 705 ttgttcagaagtatcc 2-10-4 TTgttcagaagtATCC 705_1 17625 -19 706 gatgatttgcttggag 2-10-4 GAtgatttgcttGGAG 706_1 17,646 -twenty one 707 gaaatcattcacaacc 3-10-3 GAAatcattcacaACC 707_1 17,860 -17 708 ttgtaacatctactac 3-10-3 TTGtaacatctacTAC 708_1 17,891 -16 709 cattaagcagcaagtt 3-11-2 CATtaagcagcaagTT 709_1 17,923 -17 710 ttactagatgtgagca 3-11-2 TTActagatgtgagCA 710_1 17,942 -18 711 tttactagatgtgagc 2-11-3 TTtactagatgtgAGC 711_1 17,943 -18 712 gaccaagcaccttaca 3-11-2 GACcaagcaccttaCA 712_1 17,971 -twenty two 713 agaccaagcaccttac 3-10-3 AGAccaagcacctTAC 713_1 17,972 -twenty two 714 atgggttaaataaagg 2-10-4 ATgggttaaataAAGG 714_1 18052 -15 715 tcaaccagagtattaa 2-12-2 TCaaccagagtattAA 715_1 18067 -13 716 gtcaaccagagtatta 3-11-2 GTCaaccagagtatTA 716_1 18068 -18 717 attgtaaagctgatat 2-11-3 ATtgtaaagctgaTAT 717_1 18135 -14 718 cacataattgtaaagc 2-10-4 CAcataattgtaAAGC 718_1 18141 -16 719 gaggtctgctatttac 2-11-3 GAggtctgctattTAC 719_1 18274 -19 720 tgtagattcaatgcct 2-11-3 TGtagattcaatgCCT 720_1 18404 -20 721 cctcattatactatga 2-11-3 CCtcattatactaTGA 721_1 18456 -19 722 ccttatgctatgacac 2-12-2 CCttatgctatgacAC 722_1 18509 -18 723 tccttatgctatgaca 4-10-2 TCCTtatgctatgaCA 723_1 18510 -twenty two 724 aagatgtttaagtata 3-10-3 AAGatgtttaagtATA 724_1 18598 -13 725 ctgattattaagatgt 2-10-4 CTgattattaagATGT 725_1 18607 -17 726 tggaaaggtatgaatt 2-12-2 TGgaaaggtatgaaTT 726_1 18808 -13 727 acttgaatggcttgga 2-12-2 ACttgaatggcttgGA 727_1 18880 -18 728 aacttgaatggcttgg 3-10-3 AACttgaatggctTGG 728_1 18881 -19 729 caatgtgttactattt 4-10-2 CAATgtgttactatTT 729_1 19004 -16 730 acaatgtgttactatt 3-10-3 ACAatgtgttactATT 730_1 19005 -15 731 catctgctatataaga 4-10-2 CATCtgctatataaGA 731_1 19063 -18 732 cctagagcaaatactt 4-10-2 CCTAgagcaaatacTT 732_1 19223 -20 733 cagagttaataataag 3-10-3 CAGagttaataatAAG 733_1 19327 -13 734 gttcaagcacaacgaa 4-10-2 GTTCaagcacaacgAA 734_1 19493 -18 735 agggttcaagcacaac 2-11-3 AGggttcaagcacAAC 735_1 19496 -18 736 tgttggagacactgtt 2-12-2 TGttggagacactgTT 736_1 19677 -17 737 aaggaggagttaggac 3-11-2 AAGgaggagttaggAC 737_1 19821 -18 738 ctatgccatttacgat 4-10-2 CTATgccatttacgAT 738_1 19884 -twenty one 739 tcaaatgcagaattag 2-12-2 TCaaatgcagaattAG 739_1 19913 -12 740 agtgacaatcaaatgc 2-10-4 AGtgacaatcaaATGC 740_1 19921 -18 741 aagtgacaatcaaatg 2-11-3 AAgtgacaatcaaATG 741_1 19922 -12 742 gtgtaccaagtaacaa 3-11-2 GTGtaccaagtaacAA 742_1 19978 -16 743 tgggatgttaaactga 3-10-3 TGGgatgttaaacTGA 743_1 20037 -20 The motif sequence represents the contiguous sequence of nucleobases present in the oligonucleotide. Design refers to the gapmer design FG-F', where each value represents the number of consecutive modified nucleosides (for example, 2'modified nucleosides) (first value = 5'flanking), followed by the number of DNA nucleosides (the first Second value = gap region), followed by the number of modified nucleosides (such as 2'modified nucleosides) (third value = 3'flanking), optionally followed by other repeating regions of DNA and LNA, the The iso-repeat region is not necessarily a part of the adjacent sequence complementary to the target nucleic acid. Oligonucleotide compounds represent specific designs of motif sequences. Uppercase letters represent β-D-oxyl LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages. Table 6: Oligonucleotides targeting mouse PD-L1 transcript (SEQ ID NO: 4), the design of these oligonucleotides, and specific oligonucleotide compounds designed based on the motif sequence (by CMP ID NO indication). SEQ ID NO Motif sequence design Oligonucleotide compounds CMP ID NO SEQ ID NO: The starting point on 4 dG 744 agtttacattttctgc 3-10-3 AGTttacattttcTGC 744_1 4189 -20 745 tatgtgaagaggagag 3-10-3 TATgtgaagaggaGAG 745_1 7797 -19 746 cacctttaaaacccca 3-10-3 CACctttaaaaccCCA 746_1 9221 -twenty three 747 tcctttataatcacac 3-10-3 TCCtttataatcaCAC 747_1 10386 -19 748 acggtattttcacagg 3-10-3 ACGgtattttcacAGG 748_1 12389 -twenty one 749 gacactacaatgagga 3-10-3 GACactacaatgaGGA 749_1 15088 -20 750 tggtttttaggactgt 3-10-3 TGGtttttaggacTGT 750_1 16410 -twenty one 751 cgacaaattctatcct 3-10-3 CGAcaaattctatCCT 751_1 18688 -20 752 tgatatacaatgctac 3-10-3 TGAtatacaatgcTAC 752_1 18735 -16 753 tcgttgggtaaattta 3-10-3 TCGttgggtaaatTTA 753_1 19495 -17 754 tgctttataaatggtg 3-10-3 TGCtttataaatgGTG 754_1 19880 -19 The motif sequence represents the contiguous sequence of nucleobases present in the oligonucleotide. Design refers to the gapmer design FG-F', where each value represents the number of consecutive modified nucleosides (for example, 2'modified nucleosides) (first value = 5'flanking), followed by the number of DNA nucleosides (the first Second value = gap region), followed by the number of modified nucleosides (such as 2'modified nucleosides) (third value = 3'flanking), optionally followed by other repeating regions of DNA and LNA, the The iso-repeat region is not necessarily a part of the adjacent sequence complementary to the target nucleic acid. Oligonucleotide compounds represent specific designs of motif sequences. Uppercase letters represent β-D-oxyl LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleoside linkages are phosphorothioate internucleoside linkages. Table 7: Oligonucleotide motif sequences and antisense compounds with 5'ca biocleavable linker. SEQ ID NO Motif sequence Oligonucleotide compound with ca linker CMP ID NO 755 caagtttacattttctgc c o a o AGTttacattttcTGC 755_1 756 catatgtgaagaggagag c o a o TATgtgaagaggaGAG 756_1 757 cacctttaaaacccca c o a o CACctttaaaaccCCA 757_1 758 catcctttataatcacac c o a o TCCtttataatcaCAC 758_1 759 caacggtattttcacagg c o a o ACGgtattttcacAGG 759_1 760 cagacactacaatgagga c o a o GACactacaatgaGGA 760_1 761 catggtttttaggactgt c o a o TGGtttttaggacTGT 761_1 762 cacgacaaattctatcct c o a o CGAcaaattctatCCT 762_1 763 catgatatacaatgctac c o a o TGAtatacaatgcTAC 763_1 764 catcgttgggtaaattta c o a o TCGttgggtaaatTTA 764_1 765 catgctttataaatggtg c o a o TGCtttataaatgGTG 765_1 766 caacaaataatggttactct c o a o ACAAataatggttaCTCT 766_1 767 cacagattgatggtagtt c o a o CAGAttgatggtagTT 767_1 768 cacctatttaacatcagac c o a o CCtatttaacatcAGAC 768_1 769 cactaattgtagtagtactc c o a o CTAattgtagtagtaCTC 769_1 770 caataaacatgaatctctcc c o a o ATaaacatgaatctCTCC 770_1 Uppercase letters represent β-D-oxyl LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, subscript o represents the linkage between phosphodiester nucleosides and unless otherwise indicated, other The internucleoside linkage is the phosphorothioate internucleoside linkage. Table 8: GalNAc-conjugated antisense oligonucleotide compounds. Antisense oligonucleotide conjugate CMP ID NO GN2-C6 o c o a o AGTttacattttcTGC 755_2 GN2-C6 o c o a o TATgtgaagaggaGAG 756_2 GN2-C6 o c o a o CACctttaaaaccCCA 757_2 GN2-C6 o c o a o TCCtttataatcaCAC 758_2 GN2-C6 o c o a o ACGgtattttcacAGG 759_2 GN2-C6 o c o a o GACactacaatgaGGA 760_2 GN2-C6 o c o a o TGGtttttaggacTGT 761_2 GN2-C6 o c o a o CGAcaaattctatCCT 762_2 GN2-C6 o c o a o TGAtatacaatgcTAC 763_2 GN2-C6 o c o a o TCGttgggtaaatTTA 764_2 GN2-C6 o c o a o TGCtttataaatgGTG 765_2 GN2-C6 o c o a o ACAAataatggttaCTCT 766_2 GN2-C6 o c o a o CAGAttgatggtagTT 767_2 GN2-C6 o c o a o CCtatttaacatcAGAC 768_2 GN2-C6 o c o a o CTAattgtagtagtaCTC 769_2 GN2-C6 o c o a o ATaaacatgaatctCTCC 770_2 GN2 represents the trivalent GalNAc cluster shown in Figure 3, C6 represents an aminoalkyl group with 6 carbons, uppercase letters represent β-D-oxyl LNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, subscript o represents phosphodiester nucleoside linkage and unless otherwise indicated, internucleoside linkage is phosphorothioate internucleoside linkage. Chemical schemes representing some of the molecules are shown in Figures 4 to 8. AAV/HBV mouse model Pasteur (Pasteur) Model : Generate HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout (referred to herein as HLA-A2/DR1) mice and at the Pasteur Institute (Institut Pasteur) Feeding. These mice represent in vivo experimental models used in the study of human immune function without any interference with mouse MHC response (Pajot et al., 2004 Eur J Immunol. 34(11): 3060-9). In these studies, the adeno-associated virus (AAV) vector AAV serotype 2/8 that carries the replicable HBV DNA gene body was used. Dilute the AAV-HBV vector (GVPN lot number 6163) in sterile phosphate buffered saline (PBS) to reach 5 × 1011 The titer of vg/mL. Inject 100μL of this diluted solution (dose/mouse: 5 × 1010 vg). Detect intact virus particles containing HBV DNA in the blood of HBV-carrying mice. Detect the HBcAg in the liver and the circulating HBV proteins HBeAg and HBsAg in the blood within a maximum of one year. In AAV2/8-HBV transduced mice, HBsAg, HBeAg and HBV DNA persisted in the serum for at least one year (Dion et al., 2013 J Virol 87:5554-5563). Shanghai model : In this model, mice infected with recombinant adeno-associated virus (AAV) carrying HBV gene bodies (AAV/HBV) maintained stable viremia and antigenemia for more than 30 weeks (Dan Yang et al., 2014 Cellular & Molecular Immunology 11, 71–78). Male C57BL/6 mice (4-6 weeks old) free of specific pathogens were purchased from SLAC (Shanghai Laboratory Animal Center of Chinese Academy of Sciences) and kept in animal care facilities in individual ventilated cages. Follow the animal care and use guidelines as instructed by WuXi IACUC (Institutional Animal Care and Use Committee, WUXI IACUC protocol number R20131126-mouse). The mice were adapted to the new environment for 3 days and grouped according to the experimental design. Dilute the recombinant AAV-HBV in PBS and use 200 µL for each injection. This recombinant virus carries 1.3 copies of the HBV gene body (genotype D, serotype ayw). On day 0, all mice were injected with 200 µL of AAV-HBV via the tail vein. At 6, 13, and 20 days after the AAV injection, blood was drawn from all mice (0.1 ml blood/mouse) submandibularly for serum collection. On the 22nd day after injection, mice with stable viremia were prepared for oligonucleotide therapy. Oligonucleotides can be unconjugated or GalNAc conjugated.DNA vaccine Plasmid DNA is endotoxin-free and is produced by Plasmid-Factory (Germany). pCMV-S2.S ayw encodes the preS2 and S domains of HBsAg (genotype D), and its expression is controlled by the cytomegalovirus very early gene promoter (Michel et al., 1995 Proc Natl Acad Sci USA 92:5307-5311) . pCMV-HBc encodes the HBV capsid carrying hepatitis core (HBc) Ag (Dion et al., 2013 J Virol 87:5554-5563). The treatment is performed using DNA vaccines as described herein. Five days before vaccination, the mice were injected with cardiotoxin (CaTx, Latoxan refL81-02, 50 µl/muscle). CaTx depolarizes muscle fibers to induce cell degradation. 5 days after injection, new muscle fibers appear and receive DNA vaccine to obtain better efficacy against transfection. Mix equal amounts of pCMV-S2.S ayw and pCMVCore (1 mg/ml each) and put the mice under anesthesia (100 µL 12.5 mg/mL ketamine, 1.25 mg/mL xylazine) Bilateral intramuscular injections into the cardiotoxin-treated tibialis anterior muscle received a total of 100 μg of the mixture, as previously described in Michel et al., 1995 Proc Natl Acad Sci USA 92:5307-5311.anti- PD-L1 Antibody This is a pure mouse anti-mouse PD-L1 IgG1 antibody line 6E11 produced internally at Genetech. It is a replacement antibody that cross-blocks atezolizumab and has similar in vitro blocking activity to atezolizumab produced internally at Roche. The antibody is administered by intraperitoneal (i.p.) injection at a dose of 12.5 µg/g.Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the industry. The following are applicable solutions. The oligonucleotide of the present invention can be produced by a method that slightly changes the device, carrier, and concentration used. Oligonucleotides were synthesized on Oligomaker 48 on a 1 μmol scale using phosphoramidite method on uridine universal carrier. At the end of the synthesis, the oligonucleotides were cleaved from the solid support using aqueous ammonia solution at 60°C for 5-16 hours. Oligonucleotides were purified by reverse phase HPLC (RP-HPLC) or by solid phase extraction and characterized by UPLC, and the molecular mass was further confirmed by ESI-MS.Extension of Oligonucleotides : Coupling of β-cyanide by using a 0.1 M solution of 5'-O-DMT protected amide in acetonitrile and DCI (4,5-dicyanoimidazole) (0.25 M) in acetonitrile as activator Ethyl-phosphoramidite (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz)L, NA-A (Bz), LNA-G(dmf) or LNA-T). For the final cycle, phosphoramidites with the desired modification can be used, such as a C6 linker used to connect the conjugate group or the conjugate group itself. Thiolation was performed by using hydrogenated yellow elements (0.01 M in 9:1 acetonitrile/pyridine) for the introduction of phosphorothioate linkages. It can be used for 0.02 M iodine in 7:2:1 THF/pyridine/water to introduce phosphodiester linkage. The remaining reagents are usually used for oligonucleotide synthesizers. For post-solid phase synthesis coupling, the commercially available C6 amine linker phosphamidite can be used in the final cycle of solid phase synthesis, and after deprotection and cleavage from the solid support, the amine link can be separated. Protect oligonucleotides. The conjugate is introduced by activating the functional group using standard synthetic methods. Alternatively, the conjugate moiety can be added to the oligonucleotide by using GalNAc- or GalNAc cluster phosphoramidite while still on a solid support, as described in PCT/EP2015/073331 or EP appl. NO. 15194811.4 .By RP-HPLC Purification : The crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10µ 150×10 mm column. Use 0.1 M pH 8 ammonium acetate and acetonitrile as buffers and a flow rate of 5 mL/min. The collected fractions were lyophilized to obtain the purified compound in the generally white solid form.abbreviation: DCI: 4,5-dicyanoimidazole DCM: Dichloromethane DMF: Dimethylformamide DMT: 4,4’-Dimethoxytrityl THF: Tetrahydrofuran Bz: Benzoyl Ibu: Isobutylene RP-HPLC: reversed-phase high performance liquid chromatographyT m analyze Dilute the oligonucleotide and RNA target (phosphate linkage, PO) duplex in 500 ml RNase-free water to 3 mM and mix with 500 ml 2×Tm Buffer (200mM NaCl, 0.2mM EDTA, 20mM pH 7.0 sodium phosphate) was mixed. The solution was heated to 95°C for 3 min and then it was annealed at room temperature for 30 min. The λ 40 UV/VIS spectrophotometer equipped with a Peltier temperature programmer (Peltier temperature programmer) PTP6 is used to measure the melting temperature (Tm ). The temperature was ramped up from 20°C to 95°C and then down to 25°C, and the absorption was recorded at 260 nm. Use the melting and annealing first derivative and local maximum to evaluate the double helix Tm .Tissue specific in vitro linker lysis analysis Use homogenates of relevant tissues (such as liver or kidney) and serum to perform in vitro lysis of FAM-labeled oligonucleotides with biologically cleavable linkers to be tested (such as DNA phosphodiester linkers (PO linkers)) . Collect tissue and serum samples from suitable animals (e.g. mice, monkeys, pigs, or rats) and place them in a homogenization buffer (0.5% Igepal CA-630, 25 mM pH 8.0 Tris, 100 mM pH 8.0 NaCl (1 N NaOH adjustment). Oligonucleotides were added to the tissue homogenate and serum until the concentration was 200 µg/g tissue. The sample was incubated at 37°C for 24 hours and then phenol-chloroform was used to extract the sample. In Dionex Ultimate 3000 The solution was analyzed by AIE HPLC using Dionex DNApac p-100 column and a gradient between 10mM-1 M sodium perchlorate (pH 7.5). For the standard, a fluorescence detector at 615 nm and a UV at 260 nm were used. Detector to measure the content of cleaved oligonucleotide and uncleaved oligonucleotide.S1 Nuclease cleavage analysis In S1 nuclease extract or serum, FAM-labeled oligonucleotides with S1 nuclease susceptible linkers (such as DNA phosphodiester linkers (PO linkers)) are subjected to in vitro cleavage. Use S1 nuclease to perform in vitro lysis of 100 µM oligonucleotides in nuclease buffer (60 U pr. 100 µL) for 20 minutes and 120 minutes. The enzymatic activity is stopped by adding EDTA to the buffer solution. The solution was analyzed by AIE HPLC on Dionex Ultimate 3000 using Dionex DNApac p-100 column and a gradient between 10 mM-1 M sodium perchlorate (pH 7.5). For the standard, a fluorescence detector at 615 nm and a uv detector at 260 nm were used to determine the content of cleaved and uncleaved oligonucleotides.Preparation of liver monocytes Liver cells from AAV/HBV mice were prepared as described below and according to the method (with slight modification) described by Tupin et al., 2006 Methods Enzymol 417:185-201. After the mice were euthanized, the liver was perfused with 10 ml of sterile PBS via the hepatic portal vein using a syringe with a G25 needle. When organs are whitish, harvest organs in Hank's Balanced Salt Solution (HBSS) (GIBCO® HBSS, 24020) + 5% decomplemented Fetal Calf Serum (FCS). The harvested liver was slightly compressed through a 100 μm cell strainer (BD Falcon, 352360) and the cells were suspended in 30 ml HBSS + 5% FCS. Centrifuge the cell suspension at 50 g for 5 min. The supernatant was then centrifuged at 289 g and 4°C for 10 min. After centrifugation, the supernatant was discarded and the pellet was resuspended in 15 mL of 35% isotonic Percoll solution (GE Healthcare Percoll No. 17-0891-01 diluted in RPMI 1640 (GIBCO, 31870)) at room temperature And transfer to a 15 ml tube. The cells were further centrifuged at 1360g and room temperature for 25 min. The supernatant was discarded by aspiration and the pellet containing monocytes was washed twice with HBSS + 5% FCS. In the complete medium (α-Minimal Essential Medium (Gibco, 22571), which is supplemented with 10% FCS (Hyclone, SH30066 No., Lot No. APG21570), 100 U/mL penicillin (penicillin) + 100 μg/mL streptomycin (streptomycin) + 0.3 mg/mL L-glutamic acid (Gibco, 10378), 1X optional amino acid (Gibco, 11140), 10 mM Hepes (Gibco, 15630), 1 mM sodium pyruvate (Gibco, 11360) and 50 Cells were cultured in μM β-mercaptoethanol (LKB, 1830)).Cell surface markers The cells were seeded in a U-shaped bottom 96-well plate and washed with PBS FACS (PBS containing 1% bovine serum albumin and 0.01% sodium azide). The cells were incubated with 5 μL of PBS FACS containing rat anti-mouse CD16/CD32 antibody and the yellow survival marker LD (Thermofisher, L34959) in the dark at 4°C for 10 min. Then, use 25 μL of monoclonal antibodies against NK P46 BV421 (rat Mab anti-mouse NK P46, Biolegend, 137612) and F4/80 (rat Mab anti-mouse F4/80 FITC, BD Biolegend, 123108) ( Mab) PBS FACS stained the cells in the dark at 4°C for 20 min and added two supplementary surface markers: PD1 (rat Mab anti-mouse PD1 PE, BD Biosciences, 551892) and PDL1 (rat Mab anti Mouse PDL1 BV711, Biolegend, 124319).Intracellular cytokine staining (ICS) analyze Perform ICS analysis on spleen cells and liver mononuclear cells. The cells were seeded in a U-shaped 96-well plate. The plate containing the cells was incubated overnight at 37°C in complete medium only (as a negative control) or with the peptides described in Table 9 (at a concentration of 2 μg/ml). After one hour of incubation, 2 μg/mL Brefeldin A (Sigma, B6542) was added. After overnight incubation, the cells were washed with PBS FACS and incubated with 5 μL of PBS FACS containing rat anti-mouse CD16/CD32 antibody and the yellow survival marker LD (Thermofisher, L34959) in the dark at 4°C for 10 min . Then, 25 μL of PBS FACS containing Mab was used to stain the cells in the dark at 4°C for 20 min. The mixture is composed of CD3 (hamster Mab anti-mouse CD3-PerCP, BD Biosciences, 553067), CD8 (rat Mab anti-mouse CD8-APC-H7, BD Biosciences, 560182), CD4 (rat Mab anti-mouse CD4 -PE-Cy7, BD Biosciences, 552775) and NK cell (rat Mab anti-mouse NK P46 BV421, Biolegend, 137612) monoclonal antibody composition. After washing several times, the cells were fixed and infiltrated in the dark with Cytofix/Cytoperm for 20 min at room temperature, and washed with Perm/Wash solution (BD Biosciences, 554714) at 4°C. Use against IFNγ (rat Mab anti-mouse IFNγ-APC, pure XMG1.2, BD Biosciences, 554413) and tumor necrosis factor α (TNFα) (rat Mab anti-mouse TNFα-FITC, Purified MP6-XT22; 1/250 (BD Biosciences 554418) antibody was stained with intracellular cytokines for 30 min. Before analysis by flow cytometry using the MACSQuant analyzer, the cells were washed with Perm/Wash and resuspended in In PBS FACS containing 1% formaldehyde. CD3+CD8+CD4- and cell CD3+CD8-CD4+ are selected and displayed on the dot plot. Define two regions to gate the positive cells for each cytokine. Divide the number of events found in the gates by the total number of events in the parental population to obtain the percentage of reactive T cells. For each mouse, the percentage obtained in the separate medium can be regarded as background and subtracted from the percentage obtained with peptide stimulation. The positive threshold is defined according to the experimental background, that is, the average percentage of stained cells obtained for each group in a single medium condition + two standard deviations. The percentage of cytokines that only represents at least 5 events can be considered positive. Table 9: HLA-A2/DR1 restricted epitopes contained in the HBV core protein and the mantle protein domain (S2+S) of HBsAg. protein Starting position End position sequence HLA restrictions references core 18 27 FLPSDFFPSV (SEQ ID NO: 773) A2 Bertoletti et al., Gastroenterology 1997;112:193-199 111 125 GRETVLEYLVSFGVW (SEQ ID NO: 774) DR1 Bertoletti et al., Gastroenterology 1997;112:193-199 Mantle protein (S2+S) 114 128 TTFHQTLQDPRVRGL (SEQ ID NO: 775) DR1 Pajot et al., Microbes Infect 2006;8:2783-2790. 179 194 QAGFFLLTRILTIPQS (SEQ ID NO: 776) A2 + DR1 Pajot et al., Microbes Infect 2006;8:2783-2790. 183 191 FLLTRILTI (SEQ ID NO: 777) A2 Sette et al., J Immunol 1994;153:5586-5592. 200 214 TSLNFLGGTTVCLGQ (SEQ ID NO: 778) A2 + DR1 Pajot et al., Microbes Infect 2006;8:2783-2790. 204 212 FLGGTTVCL (SEQ ID NO: 779) A2 Rehermann et al., J Exp Med 1995;181: 1047-1058. 335 343 WLSLLVPFV (SEQ ID NO: 780) A2 Nayersina et al., J Immunol 1993;150: 4659-4671. 337 357 SLLVPFVQWFVGLSPTVWLSV (SEQ ID NO: 781) A2 + DR1 Loirat et al., J Immunol 2000;165: 4748-4755 348 357 GLSPTVWLSV (SEQ ID NO: 782) A2 Loirat et al., J Immunol 2000;165: 4748-4755 370 379 SILSPFLPLL (SEQ ID NO: 783) A2 Mizukoshi et al., J Immunol 2004;173: 5863-5871. Example 1 Test in vitro efficacy 16 to 20 specific gapmers are mainly used in human PD-L1 transcripts to implement gene walking. In vitro experiments were performed on human leukemia mononuclear cell line THP1 and human non-Hodgkin's K lymphoma cell line (KARPAS-299).Cell line THP1 and Karpas-299 cell lines were originally purchased from the European Collection of Authenticated Cell Cultures (ECACC) and as recommended by the supplier at 37°C and 5% CO2 Maintain in a humidified incubator.Oligonucleotide potency THP-1 cells (3.104 in RPMI-GLutamax, 10% FBS, 1% Pen-Strep (Thermo Fisher Scientific) were added to the oligonucleotides (4-5 ul) in the 96-well circular bottom plate and the amount was 100 µl/ The final volume of the well is cultured for 6 days. Oligonucleotides are screened at a single concentration (20 µM) and at a dose range from 25 µM to 0.004 µM (1:3 dilution in water). Use MagNA according to the manufacturer’s instructions The Pure 96 Cellular RNA large-volume kit is used on the MagNA Pure 96 system (Roche Diagnostics) to extract total mRNA. For gene expression analysis, use the TaqMan RNA-to-ct 1-Step kit (Thermo Fisher Scientific) in the QuantStudio machine ( Applied Biosystems) used pre-designed Taqman primers targeting human PDL1 and ACTB (Thermo Fisher Scientific) used as an endogenous control to perform RT-qPCR. The 2(-Delta Delta C(T)) method was used to calculate the relative PD- The degree of expression of L1 mRNA and the inhibition percentage (in %) is compared with the control sample (untreated cells). Karpas-299 cells were cultured in RPMI 1640, 2 mM glutamic acid and 20% FBS (Sigma). The cells were plated in a 96-well plate at 10,000 cells/well, incubated for 24 hours, and then oligonucleotides dissolved in PBS were added. The final concentration of oligonucleotide in a single dose is 5 µM, and the final culture volume is 100 µl/well or from 50 µM, 15.8 µM, 5.0 µM, 1.58 µM, 0.5 µM, 0.158 µM, It is added in the dose response range of 0.05 µM to 0.0158 µM. The cells were harvested 3 days after the addition of the oligonucleotide compound and the PureLink Pro 96 RNA purification kit (Ambion) was used to extract RNA according to the manufacturer's instructions. Use M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion) and 100 mM dNTP group (Invitrogen, PCR grade) to synthesize cDNA according to the manufacturer's instructions. For gene expression analysis, TaqMan Fast Advanced Master Mix (2×) (Ambion) was used to perform qPCR against PD-L1 (Applied Biosystems; Hs01125299_m1) and TBP (Applied Biosystems; 432583) in a duplex setting using TaqMan primer analysis. The relative expression level of PD-L1 mRNA is shown in Table 10 in% of the control sample (cells treated with PBS). Table 10: In vitro potency of anti-PD-L1 compounds in THP1 and KARPAS-299 cell lines (from n=average of 3 experiments). The PD-L1 mRNA content was normalized to TBP in KARPAS-299 cells or ACTB in THP1 cells and displayed as control% (cells treated with PBS). CMP ID NO KARPAS-299 cells 5 µM CMP THP1 cells 20 µM CMP Compound (CMP) Starting point on SEQ ID NO 1 % mRNA of control sd % mRNA of control sd 5_1 50 1 32 11 TAattggctctacTGC 236 6_1 25 5 9 6 TCGCataagaatgaCT 371 7_1 29 2 15 5 TGaacacacagtcgCA 382 8_1 27 7 3 1 CTGaacacacagtCGC 383 9_1 twenty three 4 11 3 TCTgaacacacagtCG 384 10_1 32 3 19 6 TTCtgaacacacagTC 385 11_1 57 5 39 16 ACaagtcatgttaCTA 463 12_1 75 5 37 12 ACacaagtcatgttAC 465 13_1 twenty two 2 10 3 CTtacttagatgcTGC 495 14_1 33 4 twenty three 11 ACttacttagatgCTG 496 15_1 33 7 twenty one 6 GACttacttagatgCT 497 16_1 41 6 18 10 AGacttacttagaTGC 498 17_1 96 14 40 7 GCAggaagagactTAC 506 18_1 twenty two 2 9 3 AATAaattccgttCAGG 541 19_1 34 6 twenty one 9 GCAAataaattcCGTT 545 19_2 51 4 27 11 GCAaataaattccGTT 545 20_1 38 5 twenty three 7 AGCAaataaattcCGT 546 21_1 73 8 56 15 CAGAgcaaataaatTCC 548 22_1 83 8 65 10 TGGAcagagcaaataAAT 551 23_1 86 6 80 8 ATGGacagagcaAATA 554 24_1 44 4 30 2 CAgaatggacagaGCA 558 25_1 63 10 40 11 TTCtcagaatggacAG 562 26_1 31 1 39 5 CTGAactttgacATAG 663 27_1 60 4 56 19 AAgacaaacccagacTGA 675 28_1 36 4 34 10 TATAagacaaacccAGAC 678 29_1 40 4 28 13 TTATaagacaaaccCAGA 679 30_1 30 2 18 6 TGTTataagacaaaCCC 682 31_1 77 3 67 10 TAGAacaatggtaCTTT 708 32_1 81 17 20 14 GTAGaacaatggtaCT 710 33_1 29 5 14 8 AGGtagaacaatgGTA 712 34_1 32 1 43 20 AAGAggtagaacaATGG 714 35_1 70 4 35 13 GCatccacagtaaaTT 749 36_1 83 2 66 twenty one GAaggttatttaaTTC 773 37_1 18 2 15 5 CTAAtcgaatgcaGCA 805 38_1 64 7 35 10 TACccaatctaatCGA 813 39_1 69 1 49 13 TAGttacccaatcTAA 817 40_1 49 5 26 9 CATttagttacccAAT 821 41_1 twenty three 7 8 2 TCAtttagttaccCAA 822 42_1 twenty four 6 12 3 TTcatttagttaCCCA 823 43_1 51 7 40 5 GAATtaatttcattTAGT 829 44_1 71 9 45 3 CAGTgaggaattaATTT 837 45_1 60 5 45 17 CCAAcagtgaggAATT 842 46_1 63 1 37 15 CCCaacagtgaggAAT 843 47_1 31 3 29 12 TAtacccaacagtgAGG 846 48_1 44 3 27 0 TTatacccaacagTGAG 847 49_1 38 3 26 6 TTTatacccaacagTGA 848 50_1 20 4 7 1 CCTttatacccaaCAG 851 51_1 twenty two 3 6 2 TAACctttatacCCAA 854 52_1 28 1 29 16 AATaacctttataCCCA 855 53_1 80 11 48 10 GTAaataacctttaTA 859 54_1 54 4 37 14 ACTGtaaataacctTTAT 860 55_1 81 4 53 15 ATAtatatgcaatgAG 903 56_1 86 12 70 15 AGatatatatgcaaTG 905 57_1 56 8 27 7 GAGatatatatgcAAT 906 58_1 28 7 13 5 CCagagatatataTGC 909 59_1 88 13 69 twenty three CAATattccagagATAT 915 60_1 29 3 14 6 GCAAtattccagagATA 916 61_1 25 3 14 3 AGCaatattccagaGAT 917 62_1 29 4 17 2 CAGcaatattccAGAG 919 63_1 27 3 14 3 AATCagcaatattCCAG 921 64_1 twenty three 6 12 6 ACAAtcagcaataTTCC 923 65_1 53 9 43 15 ACtaagtagttacactTCT 957 66_1 32 5 14 6 CTAAgtagttacactTC 958 67_1 35 4 31 6 GACtaagtagttacaCTT 959 68_1 64 10 55 14 TGActaagtagtTACA 962 69_1 62 11 57 16 CTTTgactaagtagTTA 964 70_1 42 9 59 13 CTCtttgactaagTAG 967 71_1 81 6 56 12 GCTCtttgactaagTA 968 72_1 27 3 39 9 CCttaaatactgtTGAC 1060 73_1 75 5 36 7 CTtaaatactgttgAC 1060 74_1 35 6 43 13 TCCttaaatactgTTG 1062 75_1 57 4 79 25 TCTCcttaaatactgTT 1063 76_1 53 6 28 6 TAtcatagttctCCTT 1073 77_1 26 4 9 2 AGTatcatagttcTCC 1075 78_1 74 5 39 12 GAgtatcatagttCTC 1076 79_1 49 5 35 6 AGagtatcatagTTCT 1077 79_2 74 6 36 8 AGAgtatcatagtTCT 1077 80_1 19 2 19 13 CAGagtatcatagTTC 1078 81_1 twenty three 2 26 2 TTCAgagtatcataGT 1080 82_1 35 3 36 11 CTTcagagtatcATAG 1081 83_1 twenty four 6 20 7 TTCTtcagagtatcaTA 1082 84_1 20 2 16 2 TTTcttcagagtaTCAT 1083 85_1 33 4 37 10 GAGAaaggctaagTTT 1099 86_1 42 2 35 18 GAcactcttgtaCATT 1213 87_1 50 4 54 8 TGagacactcttgtaCA 1215 88_1 50 8 28 8 TGagacactcttgTAC 1216 89_1 61 4 33 6 CTttattaaactCCAT 1266 90_1 71 8 43 12 ACCAaactttattaAA 1272 91_1 62 5 42 9 AAACctctactaagTG 1288 92_1 twenty two 3 12 5 AGattaagacagtTGA 1310 93_1 46 3 ND ND AAgtaggagcaagaGGC 1475 94_1 42 4 60 twenty four AAAGtaggagcaagAGG 1476 95_1 86 15 46 10 GTtaagcagccaggAG 1806 96_1 66 6 82 27 AGggtaggatgggtAG 1842 97_1 83 19 62 36 AAGggtaggatgggTA 1843 98_1 60 9 69 5 CAAgggtaggatggGT 1844 98_2 76 13 34 7 CAagggtaggatggGT 1844 99_1 65 8 76 28 CCaagggtaggatgGG 1845 100_1 61 2 75 17 TCcaagggtaggatGG 1846 101_1 83 4 82 13 CTTCcaagggtaggAT 1848 102_1 45 3 52 14 ATCttccaagggtagGA 1849 103_1 29 2 17 7 AGaagtgatggctCATT 1936 104_1 26 3 twenty two 1 AAGaagtgatggcTCAT 1937 105_1 34 6 twenty two 2 GAAgaagtgatggcTCA 1938 106_1 41 5 twenty one 5 ATGAaatgtaaacTGGG 1955 107_1 40 8 29 6 CAATgaaatgtaaaCTGG 1956 108_1 twenty four 3 16 4 GCAAtgaaatgtaaACTG 1957 109_1 30 4 20 6 AGCAatgaaatgtaAACT 1958 110_1 44 4 34 14 GAGCaatgaaatgtAAAC 1959 111_1 18 1 13 3 TGaattcccatatcCGA 1992 112_1 69 8 35 8 AGaattatgaccaTAT 2010 113_1 77 7 38 10 AGGtaagaattatGACC 2014 114_1 97 10 56 13 TCAGgtaagaattaTGAC 2015 115_1 69 8 54 twenty one CTTCaggtaagaatTATG 2017 116_1 91 7 115 42 TCTTcaggtaagaATTA 2019 117_1 88 6 104 36 CTTCttcaggtaaGAAT 2021 118_1 85 6 118 17 TCTTcttcaggtaaGAA 2022 119_1 105 14 102 9 TCTtcttcaggtaAGA 2023 120_1 37 2 76 18 TGGtctaagagaaGAAG 2046 121_1 46 6 81 11 GTTGgtctaagagAAG 2049 122_1 74 11 64 4 AGTtggtctaagAGAA 2050 123_1 74 9 55 twenty one CAgttggtctaagAGAA 2050 124_1 65 9 95 twenty one GCAgttggtctaagagAA 2050 125_1 63 7 ND ND CAGTtggtctaagaGA 2051 126_1 65 6 ND ND GCagttggtctaagaGA 2051 127_1 67 14 104 34 GCagttggtctaaGAG 2052 128_1 twenty two 6 10 3 CTcatatcagggCAGT 2063 129_1 50 4 46 9 CACAcatgttctttaAC 2087 130_1 twenty two 4 12 12 TAAatacacacatgTTCT 2092 131_1 twenty four 2 43 28 GTAAatacacacatgTTC 2093 132_1 33 3 20 12 TGTAaatacacacaTGTT 2094 133_1 73 17 57 twenty one GATCatgtaaatacACAC 2099 134_1 47 5 28 14 AGATcatgtaaataCACA 2100 135_1 35 6 26 11 CAAAgatcatgtaaatACAC 2101 136_1 30 2 14 3 ACAAagatcatgtaaaTACA 2102 137_1 52 6 twenty four 18 GAATacaaagatcaTGTA 2108 138_1 33 5 20 6 AGAAtacaaagatcATGT 2109 139_1 37 1 twenty two 15 CAGAatacaaagatCATG 2110 140_1 85 6 53 8 GCAGaatacaaagATCA 2112 141_1 79 4 40 6 AGGCagaatacaaagAT 2114 142_1 56 2 53 20 AAGGcagaatacaaAGA 2115 143_1 28 5 20 5 ATTagtgagggacGAA 2132 144_1 26 2 twenty two 10 CAttagtgagggaCGA 2133 145_1 29 6 16 4 GAgggtgatggatTAG 2218 146_1 45 6 twenty two 5 TTaggagtaataAAGG 2241 147_1 65 7 44 9 TTAatgaatttggtTG 2263 148_1 84 8 43 10 CTttaatgaatttgGT 2265 149_1 32 0 15 3 CATGgattacaactAA 2322 150_1 33 2 20 4 TCatggattacaaCTA 2323 151_1 29 1 11 3 GTCatggattacaaCT 2324 152_1 64 2 40 9 CAttaaatctagTCAT 2335 153_1 97 8 63 twenty two GACAttaaatctagTCA 2336 154_1 92 7 ND ND AGGGacattaaatcTA 2340 155_1 35 4 25 15 CAAAgcattataaCCA 2372 156_1 34 3 twenty four 6 ACttactaggcaGAAG 2415 157_1 102 6 113 18 CAGAgttaactgtaCA 2545 158_1 102 10 103 15 CCAGagttaactgtAC 2546 159_1 88 7 95 18 GCcagagttaactgTA 2547 160_1 78 10 ND ND TGggccagagttaaCT 2550 161_1 59 5 26 5 CAgcatctatcagaCT 2576 162_1 78 8 42 10 TGAaataacatgagTCAT 2711 163_1 31 6 ND ND GTGaaataacatgAGTC 2713 164_1 18 2 11 3 TCTGtttatgtcacTG 2781 165_1 56 5 29 9 GTCTgtttatgtcaCT 2782 166_1 37 8 12 5 TGgtctgtttatGTCA 2784 167_1 39 1 19 3 TTGGtctgtttatgTC 2785 168_1 41 3 35 14 TCacccattgtttaAA 2842 169_1 18 3 14 4 TTcagcaaatatTCGT 2995 170_1 36 8 13 2 GTGtgttcagcaaATAT 2999 171_1 18 2 11 4 TCTattgttaggtATC 3053 172_1 67 4 26 12 ATtgcccatcttacTG 3118 173_1 71 2 33 9 TATtgcccatcttaCT 3119 174_1 47 4 20 5 AAatattgcccatCTT 3122 175_1 74 4 34 7 ATAaccttatcataCA 3174 176_1 98 19 44 12 TAtaaccttatcaTAC 3175 177_1 100 10 64 11 TTAtaaccttatcaTA 3176 178_1 72 38 28 5 TTTataaccttatCAT 3177 179_1 47 6 34 6 ACtgctattgctaTCT 3375 180_1 41 3 twenty three 6 AGgactgctattgCTA 3378 181_1 32 6 27 7 GAGgactgctattgCT 3379 182_1 83 1 46 20 ACgtagaataataaCA 3561 183_1 94 4 52 9 CCaagtgatataATGG 3613 184_1 49 2 16 3 TTagcagaccaaGTGA 3621 185_1 96 3 26 5 GTttagcagaccaaGT 3623 186_1 78 3 46 10 TGacagtgattataTT 3856 187_1 88 5 45 twenty one TGTCcaagatattgAC 3868 188_1 46 6 twenty three 6 GAAtatcctagatTGT 4066 189_1 79 3 45 14 CAaactgagaataTCC 4074 190_1 63 5 27 8 GCAaactgagaataTC 4075 191_1 77 9 37 11 TCCtattacaatcgTA 4214 192_1 74 10 36 9 TTCCtattacaatcGT 4,215 193_1 91 8 51 28 ACtaatgggaggatTT 4256 194_1 95 14 67 twenty four TAgttcagagaataAG 4429 195_1 86 5 47 16 TAacatatagttcAGA 4436 196_1 87 4 81 20 ATAacatatagttcAG 4437 197_1 101 6 67 20 CAtaacatatagttCA 4438 198_1 91 6 60 13 TCataacatatagtTC 4439 199_1 61 3 31 10 TAGCtcctaacaatCA 4507 200_1 79 12 49 11 CTCCaatctttgtaTA 4602 201_1 74 2 58 13 TCTCcaatctttgtAT 4603 202_1 53 3 33 10 TCtatttcagccaaTC 4708 203_1 25 4 30 9 CGGaagtcagagtGAA 4782 204_1 32 5 twenty one 7 TTAAgcatgaggaaTA 4798 205_1 34 10 26 11 TGAttgagcacctCTT 4831 206_1 81 12 62 12 GACtaattatttcgTT 4857 207_1 57 7 37 7 TGActaattatttCGT 4858 208_1 26 5 twenty one 6 GTGactaattattTCG 4859 209_1 48 3 33 13 CTGCttgaaatgtgAC 4870 210_1 32 1 34 13 CCtgcttgaaatgTGA 4871 211_1 60 5 50 19 ATcctgcttgaaATGT 4873 212_1 111 8 110 26 ATTataaatctatTCT 5027 213_1 107 1 67 12 GCtaaatactttcATC 5151 214_1 26 3 19 6 CAttgtaacataCCTA 5251 215_1 33 2 20 4 GCattgtaacatacCT 5252 216_1 89 8 53 16 TAatattgcaccaaAT 5295 217_1 25 2 29 9 GAtaatattgcacCAA 5297 218_1 27 1 27 6 AGataatattgcacCA 5298 219_1 79 6 45 11 GCcaagaagataATAT 5305 220_1 159 16 68 14 CACAgccacataaaCT 5406 221_1 90 2 72 12 TTgtaattgtggaaAC 5463 222_1 10 2 11 5 TGacttgtaattgTGG 5467 223_1 82 1 67 18 TCtaactgaaatagTC 5503 224_1 30 1 32 9 GTGgttctaactgaAA 5508 225_1 53 7 53 15 CAatatgggacttgGT 5522 226_1 44 1 33 10 ATGacaatatgggaCT 5526 227_1 49 1 41 14 TATGacaatatgggAC 5527 228_1 77 1 54 15 ATATgacaatatggGA 5528 229_1 100 3 98 29 CTtcacttaataaTTA 5552 230_1 90 12 80 19 CTGCttcacttaatAA 5555 231_1 91 0 79 twenty three AAgactgcttcacTTA 5559 232_1 49 8 77 34 GAATgccctaattaTG 5589 233_1 17 7 88 33 TGGaatgccctaatTA 5591 234_1 40 5 35 10 GCAaatgccagtagGT 5642 235_1 81 6 72 25 CTAatggaaggattTG 5673 236_1 97 17 87 25 AAtatagaacctaaTG 5683 237_1 98 4 83 twenty one GAAagaatagaatGTT 5769 238_1 93 2 102 26 ATGggtaatagattAT 5893 239_1 110 twenty four 44 14 GAaagagcacagggTG 6103 240_1 66 5 36 10 CTACatagagggaaTG 6202 241_1 70 4 34 8 GCttcctacataGAGG 6207 242_1 64 NA 33 6 TGCTtcctacatagAG 6208 243_1 30 NA 19 7 TGggcttgaaataTGT 6417 244_1 88 6 69 15 CATtatatttaagaAC 6457 245_1 8 2 5 2 TCggttatgttaTCAT 6470 246_1 18 9 12 4 CActttatctggTCGG 6482 247_1 37 2 19 5 AAAttggcacagcGTT 6505 248_1 46 12 29 8 ACCGtgacagtaaATG 6577 249_1 31 2 25 2 TGggaaccgtgacagTA 6581 250_1 17 2 twenty three 9 CCacatataggtcCTT 6597 251_1 15 6 twenty three 7 CAtattgctaccaTAC 6617 252_1 4 2 9 2 TCAtattgctaccATA 6618 253_1 65 12 85 14 CAATtgtcatatTGCT 6624 254_1 20 2 51 7 CATtcaattgtcataTTG 6626 255_1 48 8 91 41 TTTCtactgggaaTTTG 6644 256_1 11 5 twenty three 8 CAAttagtgcagcCAG 6672 257_1 43 7 62 13 GAATaatgttcttaTCC 6704 258_1 28 2 36 19 CACAaattgaataatgtTCT 6709 259_1 64 4 78 twenty two CATGcacaaattgaaTAAT 6714 260_1 53 8 104 73 ATCctgcaatttcaCAT 6832 261_1 54 5 59 14 CCaccatagctgatCA 6868 262_1 42 8 52 twenty two ACcaccatagctgaTCA 6868 263_1 68 5 118 66 CAccaccatagctgaTC 6869 264_1 40 2 73 20 TAgtcggcaccaccAT 6877 265_1 64 6 72 35 CttgtagtcggcaccAC 6880 266_1 56 4 82 35 CttgtagtcggcacCA 6881 267_1 41 5 46 twenty one CGcttgtagtcggcAC 6883 268_1 51 4 33 14 TCAataaagatcagGC 6942 269_1 61 2 49 10 TGgacttacaagaaTG 6986 270_1 45 7 40 9 ATGgacttacaagaAT 6987 271_1 51 12 36 12 GCTCaagaaattggAT 7073 272_1 17 0 14 5 TACTgtagaacatgGC 7133 273_1 15 3 11 3 GCAAttcatttgaTCT 7239 274_1 64 11 ND ND TGaagggaggagggacAC 7259 275_1 52 6 50 28 AGtggtgaagggaggAG 7265 276_1 79 7 ND ND TAgtggtgaagggaggAG 7265 277_1 81 6 ND ND AtagtggtgaagggaggAG 7265 278_1 70 9 ND ND TAgtggtgaagggagGA 7266 279_1 84 9 ND ND ATagtggtgaagggagGA 7266 280_1 40 6 64 53 TAGtggtgaagggaGG 7267 281_1 42 10 ND ND ATAgtggtgaagggaGG 7267 282_1 63 7 ND ND GAtagtggtgaagggaGG 7267 283_1 27 7 38 11 ATAGtggtgaagggAG 7268 284_1 60 twenty two ND ND GAtagtggtgaaggGAG 7268 285_1 twenty three 3 97 54 GAgatagtggtgAAGG 7271 286_1 51 6 72 19 CATGggagatagtgGT 7276 287_1 7 1 twenty one 9 ACAAataatggttaCTCT 7302 288_1 66 8 48 20 ACACacaaataatgGTTA 7306 289_1 67 6 58 20 GAGggacacacaaaTAAT 7311 290_1 46 2 50 twenty one ATATagagaggcTCAA 7390 291_1 twenty two 6 ND ND TTgatatagagaGGCT 7393 292_1 11 2 17 3 GCATttgatatagAGA 7397 293_1 70 18 44 8 TTtgcatttgataTAG 7400 294_1 30 1 30 9 CTGgaagaataggtTC 7512 295_1 53 5 42 10 ACTGgaagaataggTT 7513 296_1 56 2 41 15 TACTggaagaatagGT 7514 297_1 80 8 53 13 TGGCttatcctgtaCT 7526 298_1 73 6 52 14 ATggcttatcctGTAC 7527 299_1 75 7 89 25 TATGgcttatcctgTA 7528 300_1 52 5 50 11 GTAtggcttatccTGT 7529 301_1 27 3 31 6 ATgaatatatgccCAGT 7547 302_1 41 8 33 9 GAtgaatatatgCCCA 7549 303_1 8 2 ND ND CAAgatgaatataTGCC 7551 304_1 32 5 37 14 GACAacatcagtaTAGA 7572 305_1 28 5 30 twenty three CAAGacaacatcAGTA 7576 306_1 47 5 41 9 CACtcctagttccTTT 7601 307_1 39 6 33 7 AACactcctagttCCT 7603 308_1 68 3 42 14 TAacactcctagtTCC 7604 309_1 115 5 69 twenty two CTaacactcctagtTC 7605 310_1 97 16 57 14 TGataacataactgTG 7637 311_1 36 1 twenty three 10 CTgataacataaCTGT 7638 312_1 38 5 twenty four 5 TTTGaactcaagtgAC 7654 313_1 42 3 39 5 TCCTttacttagcTAG 7684 314_1 15 2 14 3 GAgtttggattagCTG 7764 315_1 49 28 ND ND TGggatatgacagGGA 7838 316_1 34 6 ND ND TGTGggatatgacaGG 7840 317_1 47 3 37 8 ATATggaagggataTC 7875 318_1 11 3 ND ND ACAggatatggaaGGG 7880 319_1 48 4 ND ND ATTTcaacaggatATGG 7885 320_1 18 2 16 4 GAgtaatttcaacAGG 7891 321_1 74 6 44 5 AGGGagtaatttcAACA 7893 322_1 38 5 56 28 ATTAgggagtaatTTCA 7896 323_1 66 9 32 11 CTtactattaggGAGT 7903 324_1 13 1 15 5 CAgcttactattaGGG 7906 325_1 26 4 20 9 TCAgcttactattAGG 7907 326_1 43 4 17 2 ATTtcagcttactaTTAG 7908 327_1 54 5 57 16 TTcagcttactaTTAG 7908 328_1 28 3 8 2 CAGAtttcagcttaCT 7913 329_1 43 4 37 16 GACtacaactagagGG 7930 330_1 45 12 36 10 AGACtacaactagaGG 7931 331_1 99 8 94 32 AAgactacaactagAG 7932 332_1 59 4 52 19 ATGAtttaattctagtCAAA 7982 333_1 100 2 84 twenty three TTTaattctagtcAAA 7982 771_1 91 9 60 19 GATTtaattctaGTCA 7984 334_1 74 6 50 5 TGAtttaattctaGTCA 7984 335_1 73 5 54 12 ATGAtttaattctagTCA 7984 336_1 15 1 26 3 GATGatttaattctagtCA 7984 337_1 71 twenty two 49 16 GAtttaattctaGTCA 7984 338_1 43 5 30 11 GATGatttaattctaGTC 7985 339_1 98 5 90 27 TGatttaattctagTC 7985 340_1 87 twenty one 86 2 GAGAtgatttaatTCTA 7988 341_1 92 5 85 27 GAGatgatttaatTCT 7989 342_1 7 1 7 1 CAGAttgatggtagTT 8030 343_1 7 2 twenty four 11 CTcagattgatgGTAG 8032 344_1 3 1 14 9 GTTagccctcagaTTG 8039 345_1 14 5 20 7 TGtattgttagcCCTC 8045 346_1 10 2 11 5 ACttgtattgttAGCC 8048 347_1 52 4 52 17 AGCcagtatcagggAC 8191 348_1 33 3 18 8 TTgacaatagtgGCAT 8213 349_1 7 2 13 5 ACAagtggtatctTCT 8228 350_1 63 8 44 15 AATCtactttacaaGT 8238 351_1 36 2 ND ND CAcagtagatgcctGATA 8351 352_1 twenty four 2 30 9 GAacacagtagatGCC 8356 353_1 twenty three 4 103 14 CTTGgaacacagtagAT 8359 354_1 20 2 45 2 ATAtcttggaacaCAG 8364 355_1 25 3 twenty four 6 TCTttaatatcttgGAAC 8368 356_1 39 2 41 10 TGatttctttaatatCTTG 8372 357_1 54 5 88 43 TGatgatttctttaaTATC 8375 358_1 31 4 45 27 AGGctaagtcatgaTG 8389 359_1 18 3 43 20 TTGAtgaggctaagTC 8395 360_1 6 2 11 2 CCAggattatactcTT 8439 361_1 43 5 40 14 GCcaggattataCTCT 8440 362_1 56 8 73 13 CTGccaggattataCT 8442 363_1 twenty three 1 33 7 CAGAaacttatactttaTG 8473 364_1 49 8 45 14 AAGCagaaacttaTACT 8478 365_1 39 6 37 4 GAAgcagaaacttaTACT 8478 366_1 26 4 45 13 TGGaagcagaaacttataCT 8478 367_1 twenty one 4 44 5 TGGaagcagaaacttaTAC 8479 368_1 97 4 70 twenty two AAgcagaaacttaTAC 8479 369_1 34 3 32 11 TGGaagcagaaactTATA 8480 370_1 71 7 46 19 AAGGgatattatggAG 8587 371_1 51 9 79 38 TGccggaagatttcCT 8641 372_1 45 6 52 25 ATGGattgggagtaGA 8772 373_1 27 7 30 8 AGatggattgggagTA 8774 374_1 13 3 28 6 AAGatggattgggaGT 8775 375_1 42 10 44 11 ACaagatggattGGGA 8777 375_2 41 3 45 14 ACaagatggattggGA 8777 376_1 83 9 88 32 AGAaggttcagaCTTT 8835 377_1 40 5 33 3 GCAgaaggttcagaCT 8837 377_2 28 5 20 4 GCagaaggttcagACT 8837 378_1 70 2 43 8 TGCAgaaggttcagAC 8838 379_1 twenty three 3 55 17 AGtgcagaaggttCAG 8840 379_2 51 6 41 8 AGTGcagaaggttcAG 8840 380_1 34 6 35 7 AAGTgcagaaggttCA 8841 381_1 44 11 twenty four 6 TAagtgcagaagGTTC 8842 382_1 37 5 45 9 TCtaagtgcagaAGGT 8844 383_1 75 5 147 26 CTCaggagttctactTC 8948 384_1 90 10 141 55 CTCaggagttctaCTT 8949 385_1 73 8 234 116 AtggaggtgactcaggAG 8957 386_1 33 4 42 7 ATggaggtgactcagGA 8958 387_1 twenty four 3 29 14 ATggaggtgactcAGG 8959 388_1 37 2 65 15 TAtggaggtgactcAGG 8959 389_1 50 10 81 19 ATatggaggtgactcaGG 8959 390_1 42 5 61 10 TATGgaggtgactcAG 8960 391_1 36 2 76 50 ATatggaggtgacTCAG 8960 392_1 52 6 64 6 CAtatggaggtgactcAG 8960 393_1 63 5 57 6 ATAtggaggtgacTCA 8961 394_1 53 7 64 12 CAtatggaggtgacTCA 8961 395_1 51 5 56 twenty four CAtatggaggtgACTC 8962 396_1 twenty three 3 41 34 GCatatggaggtgacTC 8962 397_1 34 3 54 10 TGcatatggaggtgacTC 8962 398_1 54 5 71 twenty four TtgcatatggaggtgacTC 8962 399_1 61 11 59 13 TttgcatatggaggtgacTC 8962 400_1 25 2 30 6 GCatatggaggtgaCT 8963 401_1 34 4 25 9 TGcatatggaggtgaCT 8963 402_1 25 4 31 20 TTGcatatggaggtgaCT 8963 403_1 51 6 37 11 TttgcatatggaggtgaCT 8963 404_1 26 1 33 5 TGCatatggaggtgAC 8964 405_1 25 2 69 19 TTGcatatggaggtGAC 8964 406_1 26 4 twenty four 4 TTTGcatatggaggtgAC 8964 407_1 19 3 20 7 TTTGcatatggaggtGA 8965 408_1 16 5 46 16 TTtgcatatggaGGTG 8966 409_1 9 2 9 6 AAgtgaagttcaaCAGC 8997 410_1 26 8 109 52 TGggaagtgaagTTCA 9002 411_1 31 5 twenty four 5 ATgggaagtgaagTTC 9003 412_1 49 9 19 10 GATGggaagtgaaGTT 9004 413_1 28 10 17 9 CTGtgatgggaagtGAA 9007 414_1 54 4 34 8 ATTgagtgaatccAAA 9119 415_1 11 1 14 2 AAttgagtgaatCCAA 9120 416_1 58 6 14 2 GATAattgagtgaaTCC 9122 417_1 5 1 16 3 GTGataattgagtGAA 9125 418_1 73 5 61 14 AAGaaaggtgcaaTAA 9155 419_1 86 6 64 13 CAagaaaggtgcAATA 9156 420_1 75 19 64 14 ACAAgaaaggtgcaAT 9157 421_1 75 8 50 13 ATttaaactcacaaAC 9171 422_1 twenty one 8 twenty three 6 CTgttaggttcaGCGA 9235 423_1 54 10 30 5 TCTGaatgaacatTTCG 9260 424_1 11 4 15 5 CTcattgaaggtTCTG 9281 425_1 87 3 52 8 CTAatctcattgaaGG 9286 426_1 95 1 85 13 CCtaatctcattgaAG 9287 427_1 31 7 twenty two 7 ACTttgatctttcAGC 9305 428_1 64 7 49 16 ACtatgcaacacttTG 9315 429_1 18 6 twenty one 3 CAAatagctttatCGG 9335 430_1 19 6 17 4 CCaaatagctttATCG 9336 431_1 35 4 27 8 TCCAaatagctttaTC 9337 432_1 75 8 43 7 GATCcaaatagcttTA 9339 433_1 67 11 32 8 ATgatccaaataGCTT 9341 434_1 53 5 43 6 TATGatccaaatagCT 9342 435_1 97 9 66 29 TAAAcagggctggGAAT 9408 436_1 58 12 44 17 ACttaaacagggCTGG 9412 437_1 58 10 30 12 ACacttaaacagGGCT 9414 438_1 87 38 41 3 GAACacttaaacAGGG 9416 439_1 70 4 59 33 AGAGaacacttaaACAG 9418 440_1 83 17 28 9 CTACagagaacaCTTA 9423 441_1 49 12 27 4 ATGctacagagaaCACT 9425 442_1 53 10 twenty four 13 ATAAatgctacagagAACA 9427 443_1 twenty three 6 20 10 AGataaatgctacaGAGA 9430 444_1 48 6 27 7 TAGAgataaatgcTACA 9434 445_1 51 3 32 8 TAGAtagagataaatGCT 9437 446_1 38 5 ND ND CAATatactagataGAGA 9445 447_1 52 3 31 1 TACAcaatatactagATAG 9448 448_1 65 6 48 11 CTAcacaatatacTAG 9452 449_1 67 9 29 2 GCTAcacaatatACTA 9453 450_1 103 17 65 15 ATATgctacacaatATAC 9455 451_1 71 13 129 twenty two TGATatgctacaCAAT 9459 452_1 19 4 9 1 ATGAtatgatatgCTAC 9464 453_1 75 10 45 twenty one GAGGagagagacaaTAAA 9495 454_1 68 6 43 10 CTAggaggagagagACA 9500 455_1 72 7 79 25 TATTctaggaggagAGA 9504 456_1 31 3 29 9 TTATattctaggagGAG 9507 457_1 38 5 62 17 GTTtatattctaGGAG 9510 458_1 15 6 15 8 TGgagtttatattcTAGG 9512 459_1 34 3 twenty one 3 CGtaccaccactcTGC 9590 460_1 41 5 55 twenty two TGAGgaaatcattcATTC 9641 461_1 81 8 47 twenty two TTTGaggaaatcatTCAT 9643 462_1 76 8 39 5 AGGCtaatcctattTG 9657 463_1 93 12 216 12 TTTAggctaatcCTAT 9660 464_1 15 6 30 9 TGCtccagtgtaccCT 9755 465_1 27 3 25 6 TAgtagtactcgATAG 9813 466_1 9 2 7 3 CTAattgtagtagtaCTC 9818 467_1 52 3 32 6 TGctaattgtagTAGT 9822 468_1 68 11 36 16 AGTGctaattgtagTA 9824 469_1 35 6 32 3 GCAAgtgctaattgTA 9827 470_1 91 9 ND ND GAGGaaatgaactaattTA 9881 471_1 92 5 ND ND CAGGaggaaatgaacTA 9886 472_1 67 5 42 6 CCctagagtcattTCC 9902 473_1 35 5 20 8 ATCttacatgatgaAGC 9925 474_1 13 1 20 5 GACacactcagatttcAG 9967 475_1 twenty four 4 20 2 AGacacactcagatttcAG 9967 476_1 25 4 twenty four 7 AAGacacactcagatttcAG 9967 477_1 26 6 19 4 AGacacactcagattTCA 9968 478_1 28 4 32 13 AAGacacactcagattTCA 9968 479_1 31 8 37 6 AAagacacactcagatTTCA 9968 480_1 63 7 51 26 GAAagacacactcagatTTC 9969 481_1 37 10 ND ND AAGAcacactcagatTTC 9969 482_1 41 4 ND ND AAAGacacactcagaTTTC 9969 483_1 19 5 48 14 TGAAagacacactcagatTT 9970 484_1 60 8 68 10 TGaaagacacactcaGATT 9971 485_1 42 8 63 twenty two TGAaagacacactcaGAT 9972 486_1 48 9 41 20 ATTGaaagacacacTCA 9975 487_1 27 6 27 12 TCattgaaagacaCACT 9977 488_1 88 13 121 33 TTCcatcattgaAAGA 9983 489_1 80 12 ND ND ATAAtaccacttaTCAT 10010 490_1 13 4 27 15 TTacttaatttcttTGGA 10055 491_1 32 5 60 twenty four TTAgaactagctttaTCA 10101 492_1 58 10 55 17 GAGgtacaaatatAGG 10171 493_1 4 1 12 3 CTTatgatacaacTTA 10384 494_1 37 6 35 5 TCttatgatacaaCTT 10385 495_1 30 0 27 6 TTCttatgatacaaCT 10386 496_1 27 8 18 3 CAgtttcttatgaTAC 10390 497_1 25 10 25 6 GCAgtttcttatgaTA 10391 498_1 77 6 72 29 TACAaatgtctattagGTT 10457 499_1 66 5 69 17 TGTAcaaatgtctatTAG 10460 500_1 27 10 20 4 AGCatcacaattagTA 10535 501_1 31 10 25 5 CTAatgatagtgaaGC 10548 502_1 twenty one 7 30 8 AGCtaatgatagtgAA 10550 503_1 35 5 39 8 ATGCcttgacatatTA 10565 504_1 64 11 79 26 CTCAagattattgACAC 10623 505_2 25 4 83 32 ACctcaagattaTTGA 10626 505_1 94 7 twenty two 6 ACCtcaagattaTTGA 10626 506_1 31 6 34 10 AACCtcaagattatTG 10627 507_1 55 6 62 17 CACAaacctcaagattaTT 10628 508_1 66 12 40 4 GTActtaattagACCT 10667 509_1 78 5 80 10 AGTActtaattagACC 10668 510_1 36 5 42 15 GTATgaggtggtaaAC 10688 511_1 40 4 48 twenty two AGgaaacagcagaAGTG 10723 512_1 27 7 13 6 GCacaacccagaggAA 10735 513_1 54 5 ND ND CAAgcacaacccagAG 10738 514_1 35 7 ND ND TTCaagcacaaccCAG 10740 515_1 49 6 52 15 AAttcaagcacaACCC 10742 516_1 72 4 106 49 TAATaattcaagcacaaCC 10743 517_1 43 4 57 twenty one ACTAataattcaaGCAC 10747 518_1 37 3 60 12 ATAAtactaataattcAAGC 10749 519_1 9 3 6 1 TAgatttgtgagGTAA 11055 520_1 59 10 31 5 AGCCttaattctccAT 11091 521_1 41 4 34 9 AATGatctagagcCTTA 11100 522_1 34 6 34 7 CTAatgatctagaGCC 11103 523_1 52 6 52 17 ACTaatgatctaGAGC 11104 524_1 60 4 54 10 CATtaacatgttctTATT 11165 525_1 57 4 55 8 ACAAgtacattaacatGTTC 11170 526_1 53 6 44 5 TTACaagtacattaaCATG 11173 527_1 54 11 49 17 GCTTtattcatgtTTAT 11195 528_1 34 7 17 5 GCTttattcatgttTA 11196 529_1 11 2 twenty one 4 AGAgctttattcatgtTT 11197 530_1 twenty two 4 33 7 ATAAgagctttattCATG 11200 531_1 30 5 32 15 CATAagagctttaTTCA 11202 532_1 77 8 twenty four 4 AGCAtaagagctTTAT 11205 533_1 8 3 15 6 TAGattgtttagtGCA 11228 534_1 4 2 10 2 GTagattgtttaGTGC 11229 535_1 41 6 33 11 GACAattctagtaGATT 11238 536_1 50 1 37 7 CTGacaattctaGTAG 11241 537_1 49 7 36 6 GCTGacaattctagTA 11242 538_1 59 2 42 11 AGgattaagatacgTA 11262 539_1 28 11 28 4 CAggattaagataCGT 11263 540_1 96 5 20 6 TCAggattaagataCG 11264 541_1 70 11 59 11 TTcaggattaagATAC 11265 542_1 53 5 28 4 AGGAagaaagtttgATTC 11308 543_1 92 13 59 12 TCAAggaagaaagtTTGA 11311 544_1 44 3 67 7 CTCAaggaagaaagTTTG 11312 545_1 43 4 32 4 TGCtcaaggaagaAAGT 11315 546_1 41 7 44 20 AATTatgctcaaggaAGA 11319 547_1 11 4 26 8 TAGGataccacattatGA 11389 548_1 25 4 26 12 CAtaatttattccattcCTC 11449 549_1 64 6 ND ND TGCAtaatttattcCAT 11454 550_1 48 17 49 7 ACTGcataatttatTCC 11456 551_1 91 10 92 15 CTAAactgcataattTATT 11458 552_1 85 8 38 9 ATaactaaactgCATA 11465 553_1 86 4 ND ND TTAttaataactaaaCTGC 11468 554_1 91 13 92 twenty one TAGTacattattaataaCT 11475 555_1 50 4 37 7 CATAactaaggacgTT 11493 556_1 41 5 30 7 TCataactaaggaCGT 11494 557_1 80 7 55 13 CGTCataactaaggAC 11496 558_1 86 3 59 11 TCgtcataactaagGA 11497 559_1 51 9 33 12 ATcgtcataactAAGG 11498 560_1 91 6 65 26 GTtagtatcttacATT 11525 561_1 30 3 41 8 CTCtattgttagtATC 11532 562_1 59 8 18 6 AGTatagagttacTGT 11567 563_1 65 11 41 11 TTCCtggtgatactTT 11644 564_1 57 13 45 13 GTTCctggtgatacTT 11645 565_1 57 15 30 7 TGttcctggtgataCT 11646 566_1 17 4 35 4 ATaaacatgaatctCTCC 11801 567_1 16 3 30 4 CTTtataaacatgaaTCTC 11804 568_1 60 5 45 11 CTGtctttataaaCATG 11810 569_1 20 2 19 5 TTgttataaatctgTCTT 11820 570_1 68 9 44 4 TTAaatttattcttgGATA 11849 571_1 76 8 48 12 CTtaaatttattctTGGA 11851 572_1 62 5 66 5 CTTCttaaatttattctTG 11853 573_1 28 4 44 10 TATGtttctcagtAAAG 11877 574_1 29 6 36 11 GAAttatctttaaACCA 11947 575_1 74 6 34 7 CCCttaaatttctaCA 11980 576_1 37 8 30 9 ACACtgctcttgtaCC 11995 577_1 45 14 27 6 TGAcaacactgctCTT 12000 578_1 2 1 12 5 TACAtttattgggcTC 12081 579_1 65 14 39 9 GTacatttattgGGCT 12082 580_1 34 4 53 12 TTGgtacatttatTGG 12085 581_1 41 7 35 6 CATGttggtacattTAT 12088 582_1 11 4 12 5 AATCatgttggtacAT 12092 583_1 96 16 48 9 AAatcatgttggtaCA 12093 584_1 71 15 42 13 GACaagtttggattAA 12132 585_1 46 34 39 6 AAtgttcagatgCCTC 12197 586_1 37 26 28 12 GCttaatgttcagaTG 12201 587_1 75 8 43 12 CGTAcatagcttgaTG 12267 588_1 41 10 28 5 GTGaggaattaggaTA 12753 589_1 41 5 27 9 GTAacaatatggttTG 12780 590_1 67 10 37 7 GAaatattgtagaCTA 13151 591_1 97 10 80 12 TTGaaatattgtagAC 13153 592_1 64 10 47 9 AAgtctagtaatTTGC 13217 593_1 84 7 60 9 GCTCagtagattatAA 13259 594_1 42 8 32 9 CATacactgttgcTAA 13296 595_1 101 6 79 17 ATGgtctcaaatcATT 13314 596_1 53 14 46 7 CAATggtctcaaatCA 13316 597_1 47 6 36 6 TTCCtattgattgaCT 13568 598_1 97 12 41 6 TTTCtgttcacaacAC 13,600 599_1 85 1 49 11 AGgaacccactaaTCT 13702 600_1 56 3 34 7 TAAatggcaggaacCC 13710 601_1 15 4 twenty four 8 GTAAatggcaggaaCC 13711 602_1 40 6 26 8 TTgtaaatggcagGAA 13713 603_1 59 12 26 6 TTatgagttaggCATG 13835 604_1 62 2 42 10 CCAggtgaaactttAA 13935 605_1 77 9 55 18 CCCttagtcagctCCT 13,997 606_1 82 13 42 11 ACccttagtcagCTCC 13998 607_1 74 1 39 10 CAcccttagtcagCTC 13,999 608_1 76 9 30 8 TCTcttactaggcTCC 14091 609_1 82 5 50 13 CCtatctgtcatcATG 14178 610_1 82 1 48 12 TCCtatctgtcatcAT 14179 611_1 41 6 50 13 GAGaagtgtgagaaGC 14,808 612_1 70 5 84 19 CATCcttgaagtttAG 14,908 613_1 64 14 61 16 TAAtaagatggctCCC 15046 614_1 85 2 51 14 CAAggcataataagAT 15053 615_1 47 1 35 10 CCaaggcataatAAGA 15054 616_1 74 8 53 11 TGatccaattctcaCC 15151 617_1 63 4 41 11 ATGatccaattctCAC 15152 618_1 46 7 42 9 CGCttcatcttcacCC 15260 619_1 104 4 15 4 TAtgacactgcaTCTT 15317 620_1 8 3 8 5 GTAtgacactgcaTCT 15318 621_1 twenty one 3 27 10 TGtatgacactgCATC 15319 622_1 37 7 38 11 TTCTcttctgtaagTC 15363 623_1 49 7 36 11 TTctacagaggaACTA 15467 624_1 47 1 32 10 ACTacagttctacAGA 15,474 625_1 78 8 69 6 TTCCcacaggtaaaTG 15561 626_1 70 7 ND ND ATTAtttgaatatactCATT 15594 627_1 73 7 49 25 TGGGaggaaattatTTG 15606 628_1 80 5 64 11 TGACtcatcttaaaTG 15621 629_1 71 6 66 19 CTGactcatcttaaAT 15622 630_1 31 6 41 6 TTTactctgactcATC 15628 631_1 88 2 68 18 TATtggaggaattaTT 15642 632_1 53 2 27 6 GTAttggaggaattAT 15643 633_1 twenty three 3 39 7 TGgtatacttctctaagTAT 15655 634_1 42 9 33 3 GATCtcttggtataCT 15666 635_1 38 1 30 16 CAgacaactctataCC 15689 636_1 10 2 19 3 AACAtcagacaacTCTA 15693 637_1 13 1 11 3 TAACatcagacaacTC 15695 638_1 14 2 27 2 TTTAacatcagacaACTC 15695 639_1 101 14 81 16 ATttaacatcagacAA 15698 640_1 14 1 17 1 CCtatttaacatcAGAC 15,700 641_1 65 2 ND ND TCCctatttaacaTCA 15703 642_1 41 6 42 12 TCAAcgactattgGAAT 15737 643_1 37 2 29 5 CTTAtattctggcTAT 15,850 644_1 31 7 35 4 ATCCttatattctgGC 15853 645_1 13 3 8 1 GAtccttatattCTGG 15854 646_1 25 5 20 4 TGAtccttatattCTG 15,855 647_1 33 6 54 10 ATTGaaacttgaTCCT 15864 648_1 43 3 27 6 ACtgtcattgaaACTT 15870 649_1 54 7 32 12 TCTtactgtcattgAA 15874 650_1 12 1 25 2 AGgatcttactgtCATT 15877 651_1 13 4 11 3 GCAaatcaactccATC 15896 652_1 10 5 16 3 GTGcaaatcaactCCA 15898 653_1 7 0 36 18 CAATtatttctttgTGC 15910 654_1 twenty one 3 31 7 TGGcaacaattattTCTT 15915 655_1 75 9 73 twenty four GCTggcaacaatTATT 15919 656_1 twenty one 6 39 6 ATCCatttctactgCC 15973 657_1 25 3 38 8 TAATatctattgattTCTA 15988 658_1 14 2 11 5 TCaatagtgtagggCA 16093 659_1 11 4 10 3 TTCaatagtgtaggGC 16094 660_1 18 1 32 12 AGGTtaattaattcaATAG 16102 661_1 33 7 25 10 CATttgtaatccCTAG 16163 661_2 64 14 31 8 CATttgtaatcccTAG 16163 662_1 48 6 34 6 ACAtttgtaatccCTA 16164 663_2 29 6 twenty three 5 AAcatttgtaatCCCT 16165 663_1 30 6 18 6 AACatttgtaatCCCT 16165 664_1 49 1 26 6 TAaatttcaagttCTG 16184 665_1 17 3 30 10 GTTtaaatttcaagTTCT 16185 666_1 twenty two 7 40 9 CCAAgtttaaatttCAAG 16189 667_1 89 11 ND ND ACCCaagtttaaaTTTC 16192 668_1 60 16 87 8 CAtacagtgacccaagTTT 16199 669_1 65 9 50 12 ACatcccatacagTGA 16208 670_1 83 8 103 4 AGcacagctctaCATC 16219 671_1 80 9 150 36 ATAtagcacagcTCTA 16223 672_1 57 14 ND ND TCCatatagcacagCT 16226 673_1 53 10 106 8 ATTtccatatagCACA 16229 674_1 78 3 96 14 TTTAtttccatatAGCA 16231 675_1 77 9 31 7 TTTatttccatatAGC 16232 676_1 32 6 ND ND AAGGagaggagatTATG 16409 677_1 32 5 twenty four 6 AGTtcttgtgttagCT 16456 678_1 19 4 17 4 GAgttcttgtgttaGC 16457 679_1 14 3 25 3 ATTaattatccatCCAC 16590 680_1 11 2 20 6 ATCaattaattatcCATC 16593 681_1 31 5 40 11 AGAatcaattaattaTCC 16596 682_1 8 3 30 10 TGagataccgtgcaTG 16656 683_1 11 3 ND ND AAtgagataccgTGCA 16658 684_1 15 3 33 10 CTGtggttaggctaAT 16,834 685_1 45 7 38 7 AagagtaagggtctgtggTT 16842 686_1 twenty four 5 ND ND GATGggttaagagTAA 16854 687_1 11 2 ND ND AGCagatgggttaaGA 16858 688_1 ND ND 51 7 TGtaaacatttgTAGC 16886 689_1 83 1 54 11 CCTgcttataaatgTA 16898 690_1 103 4 73 14 TGCCctgcttataaAT 16901 691_1 104 2 64 twenty two TCttcttagttcaaTA 16935 692_1 ND ND 60 9 TGgtttctaactACAT 16,980 693_1 ND ND 94 twenty two AGtttggtttctaaCTA 16,983 694_1 8 2 17 5 GAAtgaaacttgcCTG 17047 695_1 98 6 51 9 ATTatccttacatGAT 17173 696_1 48 4 18 4 GTacccaattatcCTT 17,180 697_1 94 2 48 9 TGTacccaattatCCT 17,181 698_1 31 5 42 13 TTgtacccaattaTCC 17,182 699_1 41 4 39 6 TTTgtacccaattaTC 17183 700_1 63 0 28 12 AGCAgcaggttataTT 17,197 701_1 99 6 43 12 TGGgaagtggtctGGG 17,292 702_1 103 2 28 5 CTGgagagtgataaTA 17322 703_1 52 6 27 9 AATGctggattacgTC 17,354 704_1 67 3 37 7 CAatgctggattaCGT 17355 705_1 36 10 80 12 TTgttcagaagtATCC 17625 706_1 19 9 47 9 GAtgatttgcttGGAG 17,646 707_1 44 NA 60 9 GAAatcattcacaACC 17,860 708_1 46 9 32 9 TTGtaacatctacTAC 17,891 709_1 56 0 79 17 CATtaagcagcaagTT 17,923 710_1 30 9 46 7 TTActagatgtgagCA 17,942 711_1 29 4 36 6 TTtactagatgtgAGC 17,943 712_1 41 13 41 6 GACcaagcaccttaCA 17,971 713_1 36 19 49 11 AGAccaagcacctTAC 17,972 714_1 30 6 34 7 ATgggttaaataAAGG 18052 715_1 70 2 twenty four 8 TCaaccagagtattAA 18067 716_1 11 4 26 8 GTCaaccagagtatTA 18068 717_1 126 56 26 6 ATtgtaaagctgaTAT 18135 718_1 73 1 42 10 CAcataattgtaAAGC 18141 719_1 twenty three 9 55 18 GAggtctgctattTAC 18274 720_1 50 1 42 11 TGtagattcaatgCCT 18404 721_1 79 3 39 10 CCtcattatactaTGA 18456 722_1 27 6 30 8 CCttatgctatgacAC 18509 723_1 26 7 50 13 TCCTtatgctatgaCA 18510 724_1 59 1 48 12 AAGatgtttaagtATA 18598 725_1 54 2 50 13 CTgattattaagATGT 18607 726_1 92 10 84 19 TGgaaaggtatgaaTT 18808 727_1 twenty four 8 61 16 ACttgaatggcttgGA 18880 728_1 8 4 51 14 AACttgaatggctTGG 18881 729_1 35 4 35 10 CAATgtgttactatTT 19004 730_1 36 9 53 11 ACAatgtgttactATT 19005 731_1 70 2 41 11 CATCtgctatataaGA 19063 732_1 38 NA 42 9 CCTAgagcaaatacTT 19223 733_1 102 15 15 4 CAGagttaataatAAG 19327 734_1 37 10 8 5 GTTCaagcacaacgAA 19493 735_1 13 1 38 11 AGggttcaagcacAAC 19496 736_1 49 NA 36 11 TGttggagacactgTT 19677 737_1 48 NA 32 10 AAGgaggagttaggAC 19821 738_1 36 NA 64 11 CTATgccatttacgAT 19884 739_1 105 19 66 19 TCaaatgcagaattAG 19913 740_1 44 NA 41 6 AGtgacaatcaaATGC 19921 741_1 107 NA 68 18 AAgtgacaatcaaATG 19922 742_1 102 4 27 6 GTGtaccaagtaacAA 19978 743_1 110 10 30 16 TGGgatgttaaacTGA 20037 Example 2-Test in vitro potency with dose response curve In the in vitro potency analysis described in Example 1, the semi-logarithmic serial dilutions (50 µM, 15.8 µM, 5.0 µM, 1.58) used in KARPAS-299 cells in PBS µM, 0.5 µM, 0.158 µM, 0.05 µM to 0.0158 µM oligonucleotides) to test the selected oligonucleotides from Table 10. Evaluate the IC 50 and maximum inhibition of oligonucleotides (residual PD-L1 performance%). EC50 calculation was performed in GraphPad Prism6. The IC50 and the maximum PD-L1 knockdown amount are shown in Table 11 (in the form of% of cells treated with control (PBS)). Table 11: Maximum inhibition (in% of saline) and EC50 in KARPAS-299 cell line. CMP ID NO Maximum inhibition ( residual PD-L1 performance% ; salt water treatment result%) EC50 (µM) Compound CMP Starting point on SEQ ID NO: 1 Avg SD Avg SD 6_1 11 3.3 0.69 0.11 TCGCataagaatgaCT 371 8_1 29 1.7 0.06 0.01 CTGaacacacagtCGC 383 9_1 19 1.7 0.23 0.02 TCTgaacacacagtCG 384 13_1 14 4.7 0.45 0.12 CTtacttagatgcTGC 495 41_1 10 1.8 0.19 0.02 TCAtttagttaccCAA 822 42_1 17 1.3 0.19 0.02 TTcatttagttaCCCA 823 58_1 twenty three 1.5 0.17 0.01 CCagagatatataTGC 909 77_1 twenty four 2.4 0.16 0.02 AGTatcatagttcTCC 1075 92_1 12 2.4 0.25 0.03 AGattaagacagtTGA 1310 111_1 3 2.0 0.27 0.03 TGaattcccatatcCGA 1992 128_1 11 1.8 0.25 0.03 CTcatatcagggCAGT 2063 151_1 16 2.7 0.28 0.05 GTCatggattacaaCT 2324 164_1 19 1.6 0.15 0.01 TCTGtttatgtcacTG 2781 166_1 36 1.7 0.11 0.02 TGgtctgtttatGTCA 2784 169_1 10 1.6 0.22 0.02 TTcagcaaatatTCGT 2995 171_1 12 2.0 0.21 0.02 TCTattgttaggtATC 3053 222_1 1 2.0 0.21 0.02 TGacttgtaattgTGG 5467 233_1 1 4.3 0.89 0.17 TGGaatgccctaatTA 5591 245_1 4 2.0 0.17 0.02 TCggttatgttaTCAT 6470 246_1 7 2.1 0.25 0.03 CActttatctggTCGG 6482 250_1 0 2.5 0.23 0.03 CCacatataggtcCTT 6597 251_1 0 2.8 0.75 0.10 CAtattgctaccaTAC 6617 252_1 3 2.2 0.19 0.02 TCAtattgctaccATA 6618 256_1 5 2.2 0.32 0.03 CAAttagtgcagcCAG 6672 272_1 1 3.2 0.69 0.10 TACTgtagaacatgGC 7133 273_1 3 2.8 0.28 0.04 GCAAttcatttgaTCT 7239 287_1 1 1.4 0.13 0.01 ACAAataatggttaCTCT 7302 292_1 2 2.1 0.21 0.02 GCATttgatatagAGA 7397 303_1 0 1.2 0.21 0.01 CAAgatgaatataTGCC 7551 314_1 3 2.1 0.39 0.04 GAgtttggattagCTG 7764 318_1 3 1.4 0.14 0.01 ACAggatatggaaGGG 7880 320_1 2 2.4 0.22 0.03 GAgtaatttcaacAGG 7891 324_1 0 2.4 0.44 0.05 CAgcttactattaGGG 7906 336_1 0 2.5 0.21 0.03 GATGatttaattctagtCA 7984 342_1 1 2.2 0.12 0.01 CAGAttgatggtagTT 8030 343_1 4 1.8 0.11 0.01 CTcagattgatgGTAG 8032 344_1 0 0.9 0.12 0.01 GTTagccctcagaTTG 8039 345_1 0 2.3 0.36 0.04 TGtattgttagcCCTC 8045 346_1 1 2.1 0.22 0.02 ACttgtattgttAGCC 8048 349_1 4 2.9 0.21 0.03 ACAagtggtatctTCT 8228 359_1 6 2.9 0.39 0.05 TTGAtgaggctaagTC 8395 360_1 0 1.7 0.18 0.02 CCAggattatactcTT 8439 374_1 5 1.7 0.33 0.03 AAGatggattgggaGT 8775 408_1 3 1.8 0.21 0.02 TTtgcatatggaGGTG 8966 409_1 0 1.8 0.21 0.02 AAgtgaagttcaaCAGC 8997 415_1 0 1.4 0.23 0.02 AAttgagtgaatCCAA 9120 417_1 7 0.9 0.15 0.01 GTGataattgagtGAA 9125 424_1 6 3.2 0.19 0.03 CTcattgaaggtTCTG 9281 429_1 5 2.5 0.48 0.05 CAAatagctttatCGG 9335 430_1 1 2.7 0.68 0.09 CCaaatagctttATCG 9336 458_1 0 4.1 0.35 0.07 TGgagtttatattcTAGG 9512 464_1 0 4.1 0.56 0.10 TGCtccagtgtaccCT 9755 466_1 1 2.1 0.21 0.02 CTAattgtagtagtaCTC 9818 474_1 0 2.4 0.27 0.03 GACacactcagatttcAG 9967 490_1 0 1.9 0.29 0.03 TTacttaatttcttTGGA 10055 493_1 3 1.8 0.20 0.02 CTTatgatacaacTTA 10384 512_1 0 3.3 0.63 0.10 GCacaacccagaggAA 10735 519_1 5 1.5 0.15 0.01 TAgatttgtgagGTAA 11055 529_1 0 2.7 0.24 0.03 AGAgctttattcatgtTT 11197 533_1 6 1.5 0.14 0.01 TAGattgtttagtGCA 11228 534_1 5 0.9 0.06 0.00 GTagattgtttaGTGC 11229 547_1 1 1.6 0.26 0.02 TAGGataccacattatGA 11389 566_1 0 3.0 0.40 0.06 ATaaacatgaatctCTCC 11801 567_1 2 2.5 0.34 0.04 CTTtataaacatgaaTCTC 11804 578_1 2 1.3 0.09 0.01 TACAtttattgggcTC 12081 582_1 1 1.6 0.20 0.02 AATCatgttggtacAT 12092 601_1 1 2.1 0.47 0.05 GTAAatggcaggaaCC 13711 619_1 4 3.4 0.44 0.08 TAtgacactgcaTCTT 15317 620_1 1 1.2 0.12 0.01 GTAtgacactgcaTCT 15318 636_1 0 1.3 0.19 0.01 AACAtcagacaacTCTA 15693 638_1 0 2.2 0.36 0.04 TAACatcagacaacTC 15695 637_1 0 2.1 0.21 0.02 TTTAacatcagacaACTC 15695 640_1 2 3.3 0.42 0.06 CCtatttaacatcAGAC 15,700 645_1 1 2.9 0.34 0.04 GAtccttatattCTGG 15854 650_1 0 2.4 0.24 0.03 AGgatcttactgtCATT 15877 651_1 4 3.4 0.33 0.05 GCAaatcaactccATC 15896 652_1 0 1.3 0.16 0.01 GTGcaaatcaactCCA 15898 653_1 4 2.0 0.09 0.01 CAATtatttctttgTGC 15910 658_1 3 1.6 0.32 0.02 TCaatagtgtagggCA 16093 659_1 5 1.4 0.20 0.01 TTCaatagtgtaggGC 16094 660_1 4 2.1 0.22 0.02 AGGTtaattaattcaATAG 16102 665_1 3 1.8 0.18 0.02 GTTtaaatttcaagTTCT 16185 678_1 3 2.1 0.43 0.04 GAgttcttgtgttaGC 16457 679_1 0 3.5 0.31 0.05 ATTaattatccatCCAC 16590 680_1 4 1.6 0.12 0.01 ATCaattaattatcCATC 16593 682_1 3 2.4 0.27 0.03 TGagataccgtgcaTG 16656 683_1 0 3.2 0.16 0.03 AAtgagataccgTGCA 16658 684_1 2 2.3 0.25 0.03 CTGtggttaggctaAT 16,834 687_1 5 1.3 0.13 0.01 AGCagatgggttaaGA 16858 694_1 0 1.7 0.16 0.02 GAAtgaaacttgcCTG 17047 706_1 15 3.6 0.27 0.06 GAtgatttgcttGGAG 17,646 716_1 10 2.1 0.15 0.02 GTCaaccagagtatTA 18068 728_1 5 1.2 0.09 0.01 AACttgaatggctTGG 18881 733_1 0 12.7 8.01 3.62 CAGagttaataatAAG 19327 734_1 0 14.6 3.49 2.39 GTTCaagcacaacgAA 19493 735_1 0 2.5 0.30 0.04 AGggttcaagcacAAC 19496 In the in vitro potency analysis described in Example 1, a 1:3 continuous aqueous solution (25 µM to 0.004 µM) was used in THP-1 to test the selected oligonucleotides from Table 6. The IC 50 and the maximum inhibition (percentage of residual PD-L1 expression) of the oligonucleotides were evaluated. EC50 calculation was performed in GraphPad Prism6. IC50 and maximum PD-L1 knockdown are shown in Table 12 (in the form of% of cells treated with control (PBS)). Table 12: Maximum inhibition (in% of saline) and EC50 in THP1 cell line. CMP ID NO Maximum inhibition ( residual PD-L1 performance % ; saline %) EC50 (µM) Compound CMP Starting point on SEQ ID NO: 1 Avg SD Avg SD 6_1 12 11.5 0.73 0.38 TCGCataagaatgaCT 371 8_1 6 5.6 0.11 0.04 CTGaacacacagtCGC 383 9_1 1 14.3 0.36 0.27 TCTgaacacacagtCG 384 13_1 2 12.4 0.49 0.31 CTtacttagatgcTGC 495 41_1 14 14.6 0.38 0.27 TCAtttagttaccCAA 822 42_1 twenty one 10.4 0.22 0.10 TTcatttagttaCCCA 823 58_1 6 19.8 0.97 0.81 CCagagatatataTGC 909 77_1 5 4.8 0.14 0.04 AGTatcatagttcTCC 1075 92_1 0 12.9 0.57 0.39 AGattaagacagtTGA 1310 128_1 15 10.1 0.23 0.13 CTcatatcagggCAGT 2063 151_1 9 14.4 0.18 0.15 GTCatggattacaaCT 2324 164_1 16 22.0 0.57 0.60 TCTGtttatgtcacTG 2781 166_1 13 11.9 0.17 0.11 TGgtctgtttatGTCA 2784 169_1 0 9.3 0.22 0.11 TTcagcaaatatTCGT 2995 171_1 11 12.9 0.28 0.20 TCTattgttaggtATC 3053 222_1 16 19.7 0.68 0.64 TGacttgtaattgTGG 5467 245_1 14 6.1 0.26 0.08 TCggttatgttaTCAT 6470 246_1 28 7.3 0.10 0.20 CActttatctggTCGG 6482 252_1 19 8.0 0.29 0.12 TCAtattgctaccATA 6618 272_1 3 9.7 0.25 0.14 TACTgtagaacatgGC 7133 314_1 13 9.6 0.31 0.15 GAgtttggattagCTG 7764 344_1 11 8.0 0.14 0.06 GTTagccctcagaTTG 8039 349_1 12 12.5 0.18 0.14 ACAagtggtatctTCT 8228 415_1 11 9.6 0.26 0.12 AAttgagtgaatCCAA 9120 493_1 15 16.5 0.48 0.34 CTTatgatacaacTTA 10384 512_1 43 14.1 0.31 0.68 GCacaacccagaggAA 10735 519_1 9 12.2 0.45 0.26 TAgatttgtgagGTAA 11055 533_1 11 13.6 0.29 0.21 TAGattgtttagtGCA 11228 534_1 9 6.5 0.09 0.03 GTagattgtttaGTGC 11229 582_1 0 12.3 0.33 0.23 AATCatgttggtacAT 12092 619_1 8 10.4 0.32 0.18 TAtgacactgcaTCTT 15317 620_1 12 24.6 1.10 1.08 GTAtgacactgcaTCT 15318 638_1 2 5.4 0.00 0.00 TAACatcagacaacTC 15695 645_1 20 29.6 1.10 1.50 GAtccttatattCTGG 15854 651_1 0 11.2 0.14 0.09 GCAaatcaactccATC 15896 658_1 11 13.8 0.48 0.32 TCaatagtgtagggCA 16093 659_1 0 8.2 0.11 0.06 TTCaatagtgtaggGC 16094 733_1 0 69.6 11.03 26.95 CAGagttaataatAAG 19327 734_1 36 16.8 2.84 2.12 GTTCaagcacaacgAA 19493 The results in Tables 7 and 8 are also shown in Figure 2 (correlated with the position of the PD-L1 mRNA precursor of SEQ ID NO: 1). It can be seen from this figure that almost all compounds have an EC50 value of less than 1 µM and a target knockdown of the PD-L1 expression level in less than 25% of the control cells (treated with saline). Example 3-In vitro efficacy and efficacy of naked and GalNAc-conjugated PD-L1 antisense oligonucleotides in poly(I:C)-induced mice and the dose of PD-L1 reduction in vivo in vitro experiments- In the reaction study, the oligonucleotides in Table 6 were used in MCP-11 cells to perform potency and efficacy tests. The same oligonucleotide and GalNAc coupling form (Table 8 CMP ID NO 755_2-765_2) were tested in vivo in poly(I:C)-induced C57BL/6J female mice for their ability to reduce PD-L1 mRNA and protein expression .In vitro analysis MCP-11 cells suspended in DMEM (Sigma catalog number D0819, supplemented with 10% horse serum, 2 mM L-glutamic acid, 0.025 mg/ml gentamicin and 1 mM sodium pyruvate) (Originally purchased from ATCC) Add 8000 cells/well to the oligonucleotide (10µl) in the 96-well round bottom plate at a final volume of 200 µl/well at 37°C and 5% CO2 Incubate in a humidified incubator for 3 days. Oligonucleotides were screened at dose range concentrations (50 µM, 15.8 µM, 5.0 µM, 1.58 µM, 0.5 µM, 0.158 µM, 0.05 µM, and 0.0158 µM). The PureLink Pro 96 RNA Purification Kit (Ambion) was used to extract total RNA according to the manufacturer's instructions. Use M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion) and 100 mM dNTP group (Invitrogen, PCR grade) to synthesize cDNA according to the manufacturer's instructions. For gene expression analysis, use TaqMan Fast Advanced Master Mix (2×) (Ambion) in a duplex setting and use TaqMan primer analysis against PD-L1 (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and Gusb (Thermo Fisher Scientific; VIC -MGB-PL Mm01197698-m1) to implement qPCR. The relative expression level of PD-L1 mRNA is shown in Table 9 as the percentage of residual PD-L1 expression in the PBS control sample (cells treated with PBS). EC50 calculation was performed in GraphPad Prism6. The EC50 and the maximum PD-L1 knockdown amount are shown in Table 13 as% of control (PBS) cells. In vivo analysis C57BL/6J female mice (20-23 g; 5 mice/group) were injected subcutaneously with 5 mg/kg of unconjugated oligonucleotide targeting mouse PD-L1 or 2.8 mg/kg of targeting mice Oligonucleotide conjugated to GalNAc of mouse PD-L1. Three days later, the mice were injected intravenously with 10 mg/kg poly(I:C) (LWM, Invivogen). Mice were sacrificed 5 h after poly(I:C) injection and liver samples were placed in RNAlater (Thermo Fisher Scientific) for RNA extraction or frozen on dry ice for protein extraction. Use PureLink Pro 96 RNA Purification Kit (Ambion) to extract total RNA from homogenized liver samples according to the manufacturer's instructions. Use M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion) and 100 mM dNTP group (Invitrogen, PCR grade) to synthesize cDNA according to the manufacturer's instructions. For gene expression analysis, use TaqMan® Fast Advanced Master Mix TaqMan Fast Advanced Master Mix (2×) (Ambion) in a duplex setting using TaqMan primers to analyze against PD-L1 mRNA (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and TBP (Thermo Fisher Scientific; VIC-MGB-PL Mm00446971_m1) to perform qPCR. The relative PD-L1 mRNA expression level is shown in Table 13 (in% of the control sample of mice injected with saline and poly(I:C)). By homogenizing the liver sample in 2 ml/100 mg tissue T-PER® Tissue Protein Extraction Reagent (Thermo Fisher Scientific) (mixed with 1× Halt Protease Inhibitor Mix, without EDTA (Thermo Fisher Scientific)) Prepare liver homogenate. Coomassie Plus (Bradford) analytical reagent (Thermo Scientific) was used to measure the protein concentration in the liver homogenate according to the manufacturer's instructions. Separate the liver homogenate (40 µg protein) on a 4-12% Bis-Tris Plus polyacrylamide gel (Thermo Fisher Scientific) in 1×MOPS running buffer and use the iBLOT dry blotting system according to the manufacturer's instructions ( Thermo Fisher Scientific) transferred to nitrocellulose membrane. Cut each print horizontally into two parts at the 64 kDa tape. After blocking in TBS containing 5% skimmed milk powder and 0.05% Tween20, the membrane and rabbit monoclonal antivinculin (Abcam catalog number ab129002, diluted 1:10000 in TBS containing 5% skimmed milk powder and 0.05% Tween20 , Upper membrane) or goat multiple strains of anti-mPD-L1 (R&D Systems catalog number AF1019, diluted 1:1000, lower membrane) were incubated overnight at 4°C. Wash the membrane in TBS containing 0.05% Tween20, and expose it to HRP-conjugated pig anti-rabbit IgG (DAKO, diluted 1:3000 in TBS containing 5% skimmed milk powder and 0.05% Tween20 at room temperature). ) Or HRP-conjugated rabbit anti-goat IgG (DAKO, diluted 1:2000) for 1 h. After washing the membrane, the reactivity was checked using ECL select (Amersham GE Healthcare). For each group of mice treated with oligonucleotides, the intensity of PD-L1/vinculin bands in mice injected with saline and poly(I:C) (control) was compared to evaluate The intensity of the PD-L1 band associated with the protein band. The results are shown in Table 13, and the western blots using pairs of naked oligonucleotides and conjugated oligonucleotides are shown in Figure 9A-E. Table 13: In vitro and in vivo efficacy of oligonucleotides targeting mouse PD-L1 CMP ID NO Compound CMP Maximum inhibition (PBS%) EC50 (µM) PD-L1 mRNA ( Control %) PD-L1 protein ( relative to control ) 744_1 AGTttacattttcTGC 9.1 0.56 86 ++ 746_1 CACctttaaaaccCCA 5.0 0.46 181 nd 747_1 TCCtttataatcaCAC 4.4 0.52 104 ++ 748_1 ACGgtattttcacAGG 1.8 0.26 102 +++ 749_1 GACactacaatgaGGA 7.6 1.21 104 nd 750_1 TGGtttttaggacTGT 12.4 0.74 84 nd 751_1 CGAcaaattctatCCT 9.9 0.69 112 nd 752_1 TGAtatacaatgcTAC 10.5 1.11 142 +++ 753_1 TCGttgggtaaatTTA 5.7 0.53 116 +++ 754_1 TGCtttataaatgGTG 5.2 0.35 98 nd 755_2 5'-GN2-C6-caAGTttacattttcTGC nd nd 58 + 757_2 5'-GN2-C6-caCACctttaaaaccCCA nd nd 62 nd 758_2 5'-GN2-C6-caTCCtttataatcaCAC nd nd 53 + 759_2 5'-GN2-C6-caACGgtattttcacAGG nd nd 66 + 760_2 5'-GN2-C6-caGACactacaatgaGGA nd nd 101 nd 761_2 5'-GN2-C6-caTGGtttttaggacTGT nd nd 99 nd 762_2 5'-GN2-C6-caCGAcaaattctatCCT nd nd 84 nd 763_2 5'-GN2-C6-caTGAtatacaatgcTAC nd nd 93 +++ 764_2 5'-GN2-C6-caTCGttgggtaaatTTA nd nd 53 + 765_2 5'-GN2-C6-caTGCtttataaatgGTG nd nd 106 nd +++: similar to the intensity of the PD-L1/vinculin band of the control; ++: weaker than the intensity of the control PD-L1/vinculin band; +: much weaker than the intensity of the PD-L1/vinculin band of the control; nd = Not determined. From the data in Table 13, it can be seen that GalNAc coupling of oligonucleotides significantly improves the reduction of PD-L1 in vivo. The decrease in mRNA is usually related to the decrease in PD-L1 protein. Except for CMP ID NO: 754_1, after oligonucleotides are coupled to GalNAc, low in vitro EC50 values usually reflect good in vivo PD-L1 mRNA reduction. Example 4-In vivo PK/PD in sorted hepatocytes and non-parenchymal cells from poly(I:C)-induced mice Hepatocytes and non-parenchymal cells isolated from self-poly(I:C) Explore the distribution of naked oligonucleotides and GalNAc-conjugated oligonucleotides and PD-L1 mRNA reduction in parenchymal cells. C57BL/6J female mice (n=3 mice/group) were injected subcutaneously with 5 mg/kg unconjugated oligonucleotide (748_1) targeting mouse PD-L1 mRNA or 7 mg/kg GalNAc conjugated Oligonucleotide (759_2). Two days later, 15 mg/kg poly(I:C) (LWM, Invivogen) was injected intraperitoneally into the mice. After poly(I:C) injection, the mice were anesthetized for 18-20 h and the liver was perfused with Hank’s balanced salt solution containing 15 mM Hepes and 0.38 mM EGTA via the vena cava at a flow rate of 7 ml/min for 5 min, and then Use collagenase solution (containing 0.17 mg/ml collagenase type 2 (Worthington 4176), 0.03% BSA, 3.2 mM CaCl2 And 1.6 g/l NaHCO3 Hank's balanced salt solution) perfused for 12 min. After perfusion, remove the liver and open the liver sac, filter the liver suspension with William E medium through a 70 µm cell filter and remove an aliquot of the cell suspension (=mixed liver cells) for subsequent use analyze. Centrifuge the remaining cell suspension at 50×g for 3 min. The supernatant was collected for subsequent purification of non-parenchymal cells. The pellets were resuspended in 25 ml of William E medium (Sigma catalog number W1878, supplemented with 1× Pen/Strep, 2 mM L-glutamic acid and 10% FBS (ATCC No. 30-2030)), and contained 90 25 ml William E medium of% percoll was mixed and the hepatocytes were pelleted by centrifugation at 50×g for 10 min. After washing twice in William E medium, the precipitated hepatocytes were resuspended in William E medium. The supernatant containing non-parenchymal cells was centrifuged at 500×g for 7 min and the cells were resuspended in 4 ml RPMI medium and centrifuged at 1800×g for 30 min through two layers of percoll (25% and 50% percoll). After collecting the non-parenchymal cells between the two percoll layers, the cells were washed and resuspended in RPMI medium. Use PureLink Pro 96 RNA Purification Kit (Ambion) according to the manufacturer's instructions to extract total RNA from purified hepatocytes, non-parenchymal cells, and total liver suspension (unfractionated liver cells). Use M-MLT reverse transcriptase, random decamer RETROscript, RNase inhibitor (Ambion) and 100 mM dNTP group (Invitrogen, PCR grade) to synthesize cDNA according to the manufacturer's instructions. For gene expression analysis, use TaqMan Fast Advanced Master Mix (2×) (Ambion) in a duplex setting and use TaqMan primers to analyze PD-L1 (Thermo Fisher Scientific; FAM-MGB Mm00452054-m1) and TBP (Thermo Fisher Scientific) ; VIC-MGB-PL Mm00446971_m1). The relative degree of PD-L1 mRNA expression is shown in Table 10 (in the form of% of control samples from mice injected with saline and poly(I:C)). The use of ELISA is carried out using the biotinylated capture probe using the sequence 5´-TACCGT-s-Bio-3' and the digoxigenin coupling detection probe using the sequence 5´- DIG-C12-S1-CCTGTG-3´ Oligonucleotide content analysis. These probes consist only of LNA with a phosphodiester backbone. The liver sample (approximately 50 mg) was homogenized in 1.4 mL MagNa pure lysis buffer (Roche catalog number 03604721001) in a 2 mL Eppendorf tube containing a 5 mm stainless steel bead. The sample was homogenized on a Retsch MM400 homogenizer (Merck Eurolab) until a uniform lysate was obtained. Incubate the sample for 30 min at room temperature. The standard was generated by spiking an unconjugated antisense oligonucleotide compound (CMP ID NO 748_1) into an untreated liver sample at a defined concentration and processing it into a sample. The spiked concentration is selected to match the expected sample oligonucleotide content (within about 10 times). Dilute the homogenized sample at least 10 times in 5 × SSCT buffer (750 mM NaCl and 75 mM sodium citrate, containing 0.05% (v/v) Tween-20, pH 7.0) and use the capture-detection solution (in 35 nM capture probe and 35 nM detection probe in 5×SSCT buffer) prepare a serial dilution of 6 times 2-fold dilution and incubate at room temperature for 30 min. The sample was transferred to a 96-well Streptavidin coated plate (Nunc catalog number 436014) and each well contained 100 µL. The plate was incubated for 1 hour at room temperature with slight agitation. Wash three times with 2 × SSCT buffer and add 100 μL diluted 1:4000 in PBST (phosphate buffered saline, containing 0.05% (v/v) Tween-20, pH 7.2, freshly prepared) to each well Anti-DIG-AP Fab fragment (Roche Applied Science, catalog number 11 093 274 910), and incubated for 1 hour at room temperature under gentle agitation. Wash three times with 2 × SSCT buffer and add 100 μL of alkaline phosphatase (AP) substrate solution (Blue Phos Substrate, KPL product code 50-88-00, new). After 30 minutes of incubation, the color intensity was measured spectrophotometrically at 615 nm under slight agitation. Export the raw data from the reader (Gen5 2.0 software) to excel format and further analyze in excel. Use GraphPad Prism 6 software and logistic 4PL regression model to generate a standard curve. Table 14: Total liver suspension, hepatocytes and non-parenchymal cells from poly(I:C) mice (n=3) treated with unconjugated oligonucleotides and GalNAc-conjugated oligonucleotides PD-L1 performance and oligonucleotide content. Cell type CMP ID no PD-L1 performance ( Saline- Poly(I:C) %) Oligonucleotide content (ng/10 5 cells ) Avg SD Avg SD Total liver cells 748_1 31 12.4 2.3 0.3 759_2 28 5.3 8.3 1.1 Hepatocyte 748_1 33 8.0 5.1 3.7 759_2 7 1.0 43.8 18.9 Non-parenchymal cells 748_1 31 10.1 2.2 0.7 759_2 66 1.6 1.7 0.9 The results showed that naked (CMP ID NO: 748_1) and coupled (CMP ID NO: 759_2) oligonucleotides reduced PD-L1 mRNA in total liver cells to the same extent. In isolated hepatocytes, the effect of coupled oligonucleotides was almost 5 times stronger than that of naked oligonucleotides, and naked oligonucleotides showed stronger effects than GalNAc-coupled oligonucleotides in non-parenchymal cells Twice the amino acid. In hepatocytes and non-parenchymal cells, the decrease in PD-L1 mRNA expression is related to the content of oligonucleotides in these cell types to a certain extent. Example 5-PD-L1 knockdown in vivo in AAV/HBV mice using naked and GalNAc-conjugated PD-L1 antisense oligonucleotides In the present study, naked or GalNAc-conjugated PD-L1 was used Antisense oligonucleotides were used to treat AAV/HBV mice, and PD-L1 mRNA expression and HBV gene expression were evaluated in the liver. Use vehicle (saline), naked PD-L1 antisense oligonucleotide (CMP ID NO 752_1, under 5 mg/kg, subcutaneously) and GalNAc PD-L1 antisense oligonucleotide ( CMP ID NO 763_2, 5-8 weeks old female HLA-A2/DR1 mice (5 animals/group) were pretreated at 7 mg/kg subcutaneously. These doses correspond to oligonuclei of equal molar concentration Glycidic acid. In week 0, with 5×1010 vg AAV-HBV transduced mice (for other details, see the description of AAV/HBV mouse model in the Materials and Methods section). From W1 to W4 after AAV-HBV transduction, mice received 4 other subcutaneous injections of PD-L1 oligonucleotide or vehicle (saline solution) and were given one week apart. Blood samples were obtained one week before transduction and one week after each injection. The mice were sacrificed two weeks after the last injection and their livers were removed after PBS perfusion. Cut the liver into smaller sections and freeze directly. In order to measure HBV gene expression, Qiagen Biorobot used the QIAamp One for all nucleic acid kit (catalog number 965672) to extract DNA from serum. The serum was diluted 1:20 in PBS and lysed in 200ul buffer AL. 100 µl. The self-set eluted DNA in 100 µl. For real-time qPCR, the TaqMan Gene Expression Master Mix (Catalog No. 4369016, Applied Biosystems) is combined with the following primers F3_core, R3_core, P3_core (Integrated DNA Technologies, each reconstructed at 100uM) by adding 1:1:0.5 Use the prepared primer mixture together: Forward (F3_core): CTG TGC CTT GGG TGG CTT T (SEQ ID NO: 784) Reverse (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA (SEQ ID NO: 785) Probe (P3_core): 56-FAM-AGC TCC AAA/ZEN/TTC TTT ATA AGG GTC GAT GTC CAT G-3IABkFQ (SEQ ID NO: 786) Use 10-fold dilution (starting from 1×109 A standard curve using HBV plastids (genotype D, GTD) was prepared from one copy/µl to 1 copy/µl and used at 5µl/reaction. For each reaction, add 10µl Gene Expression Master Mix, 4.5µl water, 0.5µl primer mix and 5µl sample or standard and run qPCR. For analysis, a standard curve is used to calculate the number of copies/ml/well. The results are shown in Table 15. The expression of PD-L1 mRNA was measured by qPCR. MRNA was extracted from frozen liver sections added to a 2ml tube containing ceramic beads (Lysing Matrix D tube, 116913500, mpbio) and 1ml Trizol. Homogenize liver slices using Precellys tissue crusher. Add 200 µl of chloroform to the homogenate, vortex and centrifuge at 4°C and 10000 rpm for 20 min. Transfer the clear phase (about 500ul) containing RNA to a new tube and add the same volume of 70% EtOH. After thorough mixing, transfer the solution to the RNeasy spin column and follow the RNeasy set of artificial RNeasy mini kit (catalog number 74104, Qiagen) (including RNA digestion RNase-free DNase Set, catalog number 79254) for further RNA extraction . At 50µl H2 Elute in O. For all samples, the final RNA concentration was measured and adjusted to 100ng/ul. QPCR was performed using the Taqman RNA-to-ct 1-step kit (catalog number 4392938, Thermo Fisher) under 7.5 μl RNA according to the manufacturer's instructions. The primer mixture used contained PD-L1_1-3 (primer numbers Mm00452054_m1, Mm03048247_m1 and Mm03048248_m1) and endogenous controls (ATCB Mm00607939_s1, CANX Mm00500330_m1, YWHAZ Mm03950126_s1 and GUSB Mm01197698_m1) Use the 2^-ddct method to analyze the data. The average value of all 4 endogenous controls was used to calculate the dct value. PD-L1 performance is relative to the average value of the endogenous control and expressed in% of saline Table 15: PD-L1 mRNA expression and HBV DNA in AAV/HBV mice (n=5) treated with unconjugated and GalNAc-conjugated oligonucleotides. CMP ID no PD-L1 mRNA expression ( % of saline) HBV DNA performance ( % of saline) Avg SD Avg SD bare 752_1 55 35 72 16 GalNAc coupling 763_2 34 3 79 9 From these results, it can be seen that naked oligonucleotides and GalNAc-conjugated oligonucleotides can reduce the expression of PD-L1 mRNA in the liver of AAV/HBV mice, and GalNAc-conjugated oligonucleotides are slightly more good. The two oligonucleotides also slightly reduced the HBV DNA in the serum. Example 6-In vivo effects on T cell responses in AAV/HBV mice In the study of the present invention, antibodies or antisense oligonucleotides targeting PD-L1 were used to treat AAV/HBV mice from Pasteur . Antisense oligonucleotides are naked antisense oligonucleotides or coupled to GalNAc. During the treatment period, the animals are immunized with DNA vaccines against HBs and HBc antigens (see Materials and Methods section) to ensure that T cells are effectively triggered by antigen-presenting cells. To evaluate how the treatment affects the cell populations in the liver and spleen and the PD-L1 performance on these populations and whether it can identify HBV-specific T cell responses.Treatment programs : The female HLA-A2/DR1 mice were treated according to the protocol below. The study was conducted in two separate sub-studies, with slightly different dosing schedules, as indicated in Tables 16 and 17 below. Administer the DNA vaccine and anti-PD-L1 antibody as described in the materials and methods section. The antisense oligonucleotides used were 5 mg/kg CMP ID NO 748_1 (naked) and 7 mg/kg CMP ID NO: 759_2 (GalNAc coupled), both of which were administered in the form of subcutaneous injection (s.c.). Table 16: Treatment plan for AAV/HBV mice using DNA vaccine and DNA vaccine + anti-PD-L1 antibody, 6 mice per group sky Vehicle (group 10) DNA vaccine (group 11) DNA vaccine + anti-PDL-1 Ab (group 13) 0 AAV/HBV 29* Animal randomization 34 Salt water + same type - Ab 41 Salt water + same type - Ab 48 Salt water + same type - Ab 50 - CaTx CaTx 55* PBS+ isotype DNA DNA+Ab 62 Salt water + same type - Ab 69 PBS+ isotype DNA DNA+Ab 76* Salt water + same type - Ab 83 Salt water + same type - Ab 97* Put to death Isotype=mouse IgG control Ab, CaTx=cardiotoxin, DNA=DNA vaccine, Ab=anti-PD-L1 Ab and *=serum collection Table 17: Treatment plan for AAV/HBV mice using DNA vaccine and DNA vaccine + naked or conjugated PD-L1 oligonucleotide (ASO), 7 mice per group sky Vehicle (group 1) DNA vaccine (group 2) DNA vaccine + PDL-1 ASO (group 7) DNA vaccine + GN-PDL-1 ASO (group 8) 0 AAV/HBV 29* Animal randomization 39 brine brine 41 brine ASO GN-ASO 46 brine brine 49 brine ASO GN-ASO 53 brine brine 55 CaTx CaTx CaTx CaTx 56 brine ASO GN-ASO 59 PBS+saline DNA+PBS DNA DNA 62* brine ASO GN-ASO 67 brine brine 70 brine ASO GN-ASO 74 PBS+saline DNA+PBS DNA DNA 77 brine ASO GN-ASO 81 brine brine 84* brine ASO GN-ASO 88 brine brine 91 brine ASO GN-ASO 102 Put to death DNA=DNA vaccine, CaTx=cardiotoxin, Ab=anti-PD-L1 Ab, ASO=naked PDL-1 oligonucleotide, GN-ASO=GalNAc-PDL-1 oligonucleotide and *=serum collection At the time of sacrifice, blood, spleen and liver mononuclear cells from each mouse in each group were collected and red blood cells were consumed (lysis buffer, BD biosciences, 555899). Liver mononuclear cells require specific preparation as described in the materials and methods section.Cell population : In the liver, cell populations are analyzed using cytometry by surface markers on liver monocytes (see Materials and Methods). Compared with the control group (ie, vehicle and DNA immunization group), the frequency of NK cells in the spleen and liver of the treated mice did not change significantly. Table 18 shows that in the liver, the number of T cells in the group treated with naked PD-L1 oligonucleotide (CMP ID NO 748_1) and GalNAc-conjugated PD-L1 oligonucleotide (CMP ID NO: 759_2) and It also shows a significant increase compared to any control group (ie, vehicle and DNA immunization group) in FIG. 10A. This increase is due to the increase in CD4+ and CD8+ T cell populations (in Table 18 and Figures 10B and 10C, respectively). Table 18: T cells in the liver after treatment (in the form of millions of cells) T cells (million) CD4+ T cells (million) CD8+ T cells (million) Avg Std Avg Std Avg Std Vehicle (group 1) 0.77 0.44 0.51 0.35 0.11 0.05 DNA vaccine (group 2) 0.90 0.24 0.58 0.16 0.16 0.08 DNA vaccine + anti-PD-L1 Ab (group 13) 1.98 0.90 1.40 0.81 0.41 0.23 Vehicle (group 10) 1.73 0.87 1.13 0.55 0.40 0.25 DNA vaccine (group 11) 1.27 0.97 0.79 0.58 0.32 0.32 DNA vaccine + PD-L1 ASO (group 7) 3.78 1.31 2.46 0.72 0.79 0.39 DNA vaccine + GN-PD-L1 ASO (group 8) 3.33 0.66 2.18 0.40 0.67 0.17 PD-L1 Performance: At the time of sacrifice, the expression of PD-L1 protein was evaluated on macrophages from the spleen and liver, B and T cells. The presence of PD-L1 antibody in the surface-labeled antibody mixture (see Materials and Methods) makes it possible to quantify PD-L1 expressing cells by cytometry. In the spleen, it was observed that there was no significant difference in the percentage of PD-L1 macrophages, B cells and CD4+ T cells between treatments. % Of CD8+ T cells expressing PD-L1 in mice treated with naked PD-L1 oligonucleotide (CMP ID NO 748_1) and GalNAc-conjugated PD-L1 oligonucleotide (CMP ID NO: 759_2) Lower than other treatments (data not shown). In the liver, PD-L1 was mainly expressed on CD8+ T cells with an average frequency of 32% and 41% in the control group (two vehicle and DNA vaccination group combinations, respectively, Figure 11A). Treatment with naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide reduced the frequency of CD8+ T cells expressing PD-L1 (see Table 19 and Figure 11A). Significant differences were also observed in the percentage of cells expressing PD-L1 for B cells and CD4+ T cells after ASO treatment, but these cell types showed significantly less performance than PD-L1 for CD8+ T cells (see Table 19 and Figures 11B and C) . Treatment with anti-PD-L1 Ab also significantly reduced PD-L1 expression in all cell types. However, this reduction may be due to the PD-L1 epitope being partially blocked by the anti-PD-L1 antibody used for treatment, thereby preventing the PD-L1 detection antibody in the surface-labeled antibody mixture from preventing binding to PD-L1. Therefore, the down-regulation of PD-L1 achieved by the anti-PD-L1 antibody used for therapy seems to be the result of epitope competition between the therapeutic antibody and the detection antibody. Table 19:% of liver cell population with PD-L1 performance % Of CD8+ T cells % Of CD4+ T cells % Of B cells Avg Std Avg Std Avg Std Vehicle (group 10) 35.5 4.7 0.75 0.52 5.9 1.5 DNA vaccine (group 11) 36.8 7.7 0.61 0.08 5.5 1.1 DNA vaccine + anti-PD-L1 Ab (group 13) 18.6 12.3 0.33 0.10 2.9 1.7 Vehicle (group 1) 28.5 11.5 0.64 0.21 5.9 1.7 DNA vaccine (group 2) 44.9 14.4 1.43 0.69 8.7 3.1 DNA vaccine + PD-L1 ASO (group 7) 9.6 2.4 0.37 0.21 2.9 0.8 DNA vaccine + GN-PD-L1 ASO (group 8) 14.6 3.3 0.31 0.11 2.8 0.8 HBV Specificity T Cellular response: Use the intracellular interleukin staining analysis (see Materials and Methods section) to detect the production of IFNγ and TNFα to detect NK cells and CD4+ and CD8+ T cells that produce pro-inflammatory cytokines. In the spleen, at the time of sacrifice, no NK cells secreting IFNγ- and TNFα were detected and a small amount of CD4+ T cells could be detected (frequency <0.1%). IFNγ-producing CD8+ T cells targeting two HBV antigens were detected in mice treated with naked PD-L1 oligonucleotides or GalNAc PD-L1 oligonucleotides and mice receiving only DNA vaccines in this study ( Data not shown). In the livers of DNA-immunized HBV-carrying mice, no IFNγ-producing NK cells were detected at the time of sacrifice, while in the livers of a small number of DNA-immunized mice, specific for core or S2+S was detected at a low frequency Sexual IFNγ-producing CD4+ T cells (<0.4%, data not shown). IFNγ-producing HBV S2+S-specific CD8+ T cells were detected in most of the DNA-immunized mice. In mice treated with a combination of DNA vaccine and naked PD-L1 oligonucleotide or GalNAc PD-L1 oligonucleotide, the frequency of CD8+ T cells that secrete IFNγ increased, while the use of anti-PD-L1 antibody The treatment did not add any significant additional effects to DNA vaccination (Figure 12). IFNγ-producing CD8+ T cells targeting mantle protein and core antigen were detected in most DNA immunization groups (except anti-PD-L1 antibody) (Figure 12B). Most S2-S-specific T cells produce IFNγ and TNFα (Figure 12C). The results are also shown in Table 20. Table 20:% of HBV antigen (S2-S or core) specific CD8+ T cells from the total IFNγ or IFNγ + TNFα cell population PreS2-S specific T cells (% of IFNγ cells) Core specific T cells (% of IFNγ cells) S2-S specific T cells (IFNγ + TNFα%) Avg Std Avg Std Avg Std Vehicle (group 10) 0.15 0.37 0.18 0.43 0.00 0.00 DNA vaccine (group 11) 1.48 1.10 0.47 0.53 0.42 1.02 DNA vaccine + anti-PDL-1 Ab 1.18 0.95 0 0 0.38 0.49 Vehicle (group 1) 0.17 0.45 0.11 0.28 0.00 0.00 DNA vaccine (group 2) 1.70 1.02 0.27 0.51 0.98 0.90 DNA vaccine + PDL-1 ASO 2.56 1.60 0.78 0.80 1.44 1.55 DNA vaccine + GN-PDL-1 ASO 3.83 2.18 0.68 1.16 2.62 1.62 Example 7-In vivo effect on HBV antigen and HBV DNA in the serum of AAV/HBV mice In the study of the present invention, GalNAc-conjugated PD-L1 antisense oligonucleotide CMP ID NO 759_2 was used to treat AAV from Shanghai /HBV mice (see materials and methods section). To evaluate how the treatment affects the serum levels of HBe and HBs antigens and HBV DNA compared with vehicle-treated animals.Treatment programs : In this study, male C57BL/6 mice infected with recombinant adeno-associated virus (AAV) carrying HBV genome (AAV/HBV) were used as described in the Shanghai model in the Materials and Methods section. Mice (6 mice/group) were injected with 5 mg/kg antisense oligonucleotide CMP ID NO: 759_2 or vehicle (saline) once a week for 8 weeks, both of which were subcutaneous injection (sc) Invested. Blood samples were collected every week during treatment and 6 weeks after treatment. Measure the content of HBV DNA, HBsAg and HBeAg in serum samples as described below. The results of the first 10 weeks are shown in Table 21 and Figure 13. The research is still ongoing at the time of archiving, therefore, the remaining 4 weeks of data are not yet available.HBsAg and HBeAg Detection: Use HBsAg chemiluminescence immunoassay (CLIA) and HBeAg CLIA kit (Autobio diagnostics Co. Ltd., Zhengzhou, China, catalog numbers CL0310-2 and CL0312-2, respectively) in infected AAV-HBV mice according to the manufacturer The protocol is to determine the serum HBsAg and HBeAg content in the serum. Briefly, 50 μl of serum was transferred to the respective antibody-coated microtiter plate and 50 μl of enzyme conjugate reagent was added. The plate was incubated on a shaker at room temperature for 60 min, and then all wells were washed 6 times with a washing buffer using an automatic washer. Add 25 μl of Substrate A and then 25 μl of Substrate B to each well. The plate was incubated at room temperature for 10 minutes, and then the luminescence was measured using an Envision luminescence reader. HBsAg is given in units of IU/ml; where 1 ng HBsAg = 1.14 IU. HBeAg is given in units of NCU/ml serum.HBV DNA Extraction and qPCR : First, the mouse serum was diluted 10-fold (1:10) using phosphate buffered saline (PBS). Use MagNA Pure 96 (Roche) robot to extract DNA. 50 μl of diluted serum was mixed with 200 ul of MagNA Pure 96 external lysis buffer (Roche, catalog number 06374913001) in the treatment column and incubated for 10 minutes. Then use "MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit" (Roche, catalog number 06543588001) and "Viral NA Plasma SV external lysis 2.0" protocol to extract DNA. The DNA elution volume is 50 μl. A Taqman qPCR machine (ViiA7, life technologies) was used to quantify the extracted HBV DNA. Each DNA sample was tested in PCR in duplicate. Add 5μl DNA sample to a 384-well plate containing 10μl TaqMan Gene Expression Master Mix (Applied Biosystems, catalog number 4369016), 0.5μl PrimeTime XL qPCR primer/probe (IDT) and 4.5μl distilled water to 15μl PCR standard mix And the following settings were used to perform PCR: UDG incubation (2min, 50°C), enzyme activation (10min, 95°C) and PCR (40 cycles, with denaturation at 95°C for 15sec and annealing and extension at 60°C for 1min). Since Ct The value is based on the HBV plastid DNA standard curve and the DNA copy number is calculated by ViiA7 software. Sequence for TaqMan primer and probe (IDT): Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT T (SEQ ID NO: 784) Reverse primer (R3_core): AAG GAA AGA AGT CAG AAG GCA AAA (SEQ ID NO: 785) Taqman probe (P3_core): 56-FAM/AGC TCC AAA /ZEN/TTC TTT ATA AGG GTC GAT GTC CAT G/3IABkFQ (SEQ ID NO: 786). Table 21: The levels of HBV-DNA, HBsAg and HBeAg in blood from AAV/HBV mice after treatment with GalNAc-conjugated PD-L1 antisense oligonucleotides. brine CMP ID NO: 759_2, at 5 mg / kg at HBV-DNA HBsAg HBeAg HBV-DNA HBsAg HBeAg Days Avg Std Avg Std Avg Std Avg Std Avg Std Avg Std 0 7.46 0.35 3.96 0.48 3.23 0.14 7.44 0.29 3.87 0.40 3.17 0.13 7 7.53 0.23 4.17 0.45 3.35 0.10 7.53 0.20 3.91 0.42 3.19 0.18 14 7.57 0.24 4.12 0.49 3.19 0.11 7.45 0.22 3.90 0.50 2.99 0.27 twenty one 7.47 0.27 3.93 0.51 3.12 0.05 7.33 0.47 3.71 0.76 2.78 0.26 28 7.68 0.26 3.88 0.67 3.18 0.13 7.45 0.46 3.65 0.93 2.67 0.38 35 7.69 0.21 4.03 0.54 2.95 0.08 7.13 0.75 2.98 1.05 2.04 0.38 42 7.58 0.23 3.89 0.65 3.34 0.10 6.69 0.89 2.60 1.05 1.98 0.45 49 7.77 0.17 3.54 1.06 3.08 0.26 6.56 1.26 2.19 0.70 1.47 0.37 56 7.71 0.24 3.99 0.86 3.28 0.05 6.21 1.48 2.28 0.84 1.38 0.30 63 7.59 0.28 3.67 1.07 3.25 0.13 6.08 1.39 2.08 0.71 1.35 0.30 From this study, it can be seen that after 6 weeks of treatment, GalNAc-conjugated PD-L1 antisense oligonucleotide CMP NO 759_2 has a significant effect on the reduction of serum HBV-DNAH, BsAg and HBeAg levels, and after treatment The effect that lasted for at least 2 weeks has ceased. Example 8-In vitro PD-L1 knockdown in human primary hepatocytes using GalNAc-conjugated PD-L1 oligonucleotides Use genomics to explore the reduction of GalNAc-conjugated PD-L1 antisense oligonucleotide compounds The ability of PD-L1 transcription in primary human hepatocytes.Cell culture Refrigerated human hepatocytes are stored at approximately 5 × 106 The density of cells/ml is suspended in WME supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (0.1 mg/ml) and L-glutamic acid (0.292 mg/ml) and To 2 × 105 The density of cells/well was seeded into collagen-coated 24-well plates (Becton Dickinson AG, Allschwil, Switzerland). Before starting to process the oligonucleotides at a final concentration of 100 µM, the cells were pre-incubated for 4 h to allow connection to the cell culture plate. The oligonucleotides used are shown in Table 21 and Table 8, and the vehicle was PBS. Change the inoculation medium to 315 µl serum-free WME (supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), L-glutamic acid (0.292 mg/ml)) and 35 µl in PBS The stock solution of 1 mM oligonucleotides in the medium is added to the cell culture and left on the cells for 24 hours or 66 hours.Library preparation Using Illumina Stranded mRNA chemistry on the Illumina sequencing platform, a sequencing strategy (Q squared EA) with 2 × 51 bp paired-end reads and a minimum read depth of 30M in each sample was used to describe the transcript performance. Cells were lysed in the wells by adding 350 µl Qiagen RLT buffer and registered in the randomization protocol. Purify mRNA using Qiagen RNeasy Mini Kit. The mRNA was quantified and the integrity was assessed using an Agilent bioanalyzer. After the initial quality evaluation of the isolated RNA, it was observed that all samples met the input quality metric of 100 ng and had a RIN score> 7.0. The Illumina TruSeq Stranded mRNA library was prepared from 100 ng total RNA to generate sequencing libraries for all samples. The size distribution of the final cDNA library was analyzed using an Agilent bioanalyzer (DNA 1000 kit), quantified by qPCR (KAPA Library Quant kit) and normalized to 2 nM during preparation for sequencing. The standard cluster generation set v5 was used to bind the cDNA library to the surface of the flow cell in an isothermal manner and cBot to amplify the ligated cDNA constructs to a pure cluster of up to about 1000 copies each. The TruSeq SBS kit is used to determine DNA sequence by sequencing by synthesis technology.data processing Use the GSNAP short-read comparison program to locate the 2×51 bp Illumina paired-end sequencing read on the human reference genome hg19. Use SAMTOOLS program to compare and convert SAM format into BAM format files. The gene read count of PD-L1 was estimated based on exon annotations from NCBI RefSeq (specified by the corresponding GTF file of hg19). Use the DESeq2 R kit to apply a normalization step that takes into account the different library sizes of each sample. The reduction in PD-L1 transcription after incubation with GalNAc-conjugated PD-L1 antisense oligonucleotide compounds is shown in Table 22. Table 22: PD-L1 transcription reduction in primary primary hepatocytes after treatment with GalNAc-conjugated oligonucleotides, n=4 Compound PD-L1 performance level , 24 h ( count of library size adjustment) PD-L1 performance level , 66 h ( count of library size adjustment) Vehicle 259 156 159 168 192 136 202 211 767_2 7 7 11 14 twenty two 9 28 15 766_2 16 13 15 10 17 11 29 13 769_2 15 twenty one 18 18 25 18 26 25 768_2 41 25 27 48 31 25 34 twenty two 770_2 twenty one 16 44 62 67 51 38 63 After 24 and 66 hours of incubation, all five GalNAc-conjugated antisense compounds showed a significant reduction in PD-L1 transcription when compared with the vehicle-treated samples. Example 9-EC50 of conjugated and naked PD-L1 antisense oligonucleotides in HBV-infected ASGPR-HepaRG cells Compare two naked and equivalent GalNAc-conjugated PD-L1 antisense in HBV-infected ASGPR-HepaRG cells The efficacy of sense oligonucleotides.Cell line HepaRG cells (Biopredic International, Saint-Gregoire, France) were cultured in William E medium (supplemented with 10% HepaRG growth supplement (Biopredic)). Since this cell line, the lentiviral method was used to generate a HepaRG cell line that stably overexpresses human ASGPR1 and ASGPR2. Use the lentivirus encoding human ASGPR1 and 2 (CLV-CMV-ASGPR1-T2a_ASGPR2-IRES-Puro) produced by Sirion biotech on-demand with MOI 300 to transgenerate HepaRG under the control of the CMV promoter and the puromycin resistance gene cell. 1μg/ml puromycin was used to select transduced cells for 11 days and then maintained in the same concentration of antibiotics to ensure stable performance of the transgene. At the mRNA level, RT-qPCR (ASGPR1: 8560 times for non-transduction, ASGPR2: 2389 times for non-transduction) and flow cytometry analysis at the protein level to confirm the overexpression of ASGPR1/2. Use 1.8% DMSO to differentiate cells for at least 2 weeks before infection. HBV genotype D is derived from HepG2.2.15 cell culture supernatant and concentrated using PEG precipitation. To evaluate the activity of the test compound against HBV, differentiated ASGPR-HepaRG cells in 96-well plates were infected with HBV at an MOI of 20 to 30 for 20 h, and then the cells were washed 4 times with PBS to remove the HBV inoculum.Oligonucleotide efficacy The following oligonucleotides Naked PD-L1 ASO Equivalent GalNAc coupled to PD-L1 ASO CPM ID NO: 640_1 CPM ID NO: 768_2 CPM ID NO: 466_1 CPM ID NO: 769_2 On the 7th and 10th day after infection, a serial dilution of 25 µM to 0.4 nM (diluted 1:4 in PBS) was added to the ASGPR-HepaRG cells infected with HBV. The cells were harvested on the 13th day after infection. Use the MagNA Pure 96 Cellular RNA large-volume kit on the MagNA Pure 96 system (Roche Diagnostics) according to the manufacturer's instructions to extract total mRNA. For gene expression analysis, RT-qPCR was performed as described in Example 5. Use the 2^-ddct method to analyze the data. Actin B was used as an endogenous control to calculate the dct value. PD-L1 performance is relative to the endogenous control and saline vehicle. The EC50 calculation was implemented in GraphPad Prism6 and is shown in Table 23. Table 23: EC50 in ASGPR-HepaRG HBV infected cells, n=4. CMP ID NO EC50 (µM) 640_1 2.25 768_2 0.10 466_1 5.82 769_2 0.13 These data clearly show that the GalNAc conjugate of PD-L1 antisense oligonucleotide significantly improves the EC50 value. Example 10-Stimulation of T cell function in PBMC derived from chronic HBV patients to explore whether naked PD-L1 antisense compounds can increase chronic infection of HBV (CHB) patients after stimulation with isolated HBV antigens of peripheral blood mononuclear cells (PBMC) The T cell function. Frozen PBMC from three patients with chronic HBV infection were thawed and seeded in 100 µl of medium (RPMI1640 + GlutaMax + 8% human serum + 25 mM Hepes + 1% PenStrep) at a density of 200'000 cells/well. On the second day, use 1µM PepMix HBV large mantle protein or 1µM PepMix HBV core protein (see Table 9) with or without 5µM CMP ID NO: 466_1 or CMP ID NO: 640_1 containing 100pg/ml IL-12 and 5ng in 100µl Cells were stimulated in the medium of IL-7/ml (Only Concanavalin stimulation was applied on the 8th day). After 4 days, the PD-L1 antisense oligonucleotide treatment was renewed with a medium containing 50 IU IL-2. On the 8th day after the first stimulation, the cells were stimulated with PepMix or 5μg/ml concanavalin A and PD-L1 antisense oligonucleotides for another 24 hours. For the last 5h stimulation, add 0.1 µl Brefeldin A (Brefeldin A), 0.1 µl monensin and 3 µl anti-human CD-107 (APC). After 24h, wash the cells with staining buffer (PBS + 1% BSA + 0.09% sodium azide + EDTA) and apply surface staining at 4°C for 30 min [anti-human CD3 (BV 605), anti-human CD4 (FITC) , Anti-human CD8 (BV711), anti-human PDL1 (BV421), anti-human PD1 (PerCP-Cy5.5) and live and dead stain (BV510) (BD Biosciences)]. The cells were fixed in BD fixation buffer at 4°C for 15 min. The next morning, the cells were permeated with BD Perm/Wash buffer at 4°C for 15min and intracellular staining was performed at 4°C for 30mi [anti-human INF( (PE)]. After washing in Perm/Wash buffer, the The cells are dissolved in 250µl of staining buffer. FACS measurement was performed on BD Fortessa (BD Biosciences). For analysis, the entire cell population was first gated on live cells (live and dead stain, BV510), and then on CD3+ (BV605) cells. The CD3+ cells were then mapped as CD107a+ (APC) vs. IFNγ+ (PE). The results are shown in Table 24. Table 24: Effect of PD-L1 ASO treatment on CD3+ T cells from PBMC isolated from three patients with chronic HBV infection. No antigen stimulation Mantle protein antigen Core antigen brine CMP 466_1 CMP 640_1 brine CMP 466_1 CMP 640_1 brine CMP 466_1 CMP 640_1 INFγ-/ CD107+ 1.16 4.95 4.81 4.7 9.12 8.62 3.84 9.66 7.31 2.7 3.59 2.74 2.57 3.69 3.2 3.25 3.34 2.92 3 3.87 3.98 4.59 12.5 10.9 9.23 6.11 6.88 INFγ+/ CD107+ 0.12 1.03 1.15 3.19 17.3 18.9 2.38 15.1 5.75 0.49 3.12 1.75 2.73 7 5.34 1.63 2.35 1.9 0.24 1.13 1.5 1.6 8.16 3.06 1.68 1.9 1.91 INFγ+/ CD107- 0.33 1.43 1.08 5.11 7.74 9.47 3.14 7.76 2.83 0.61 2.9 2.26 7.84 5.79 5.78 2.33 2.82 2.95 0.17 1.57 1.72 1.22 2.58 0.99 0.1 0.61 1.04 From these data, it can be seen that antigen stimulation itself can induce T cell activation in PBMC of CHB patients (n=3) (increase the% of CD3+ cells expressing INFγ (and/or CD107a). Add PD-L1 antisense oligo Nucleotide CMP 466_1 or 640_1 made an additional increase in CD3+ T cell response. This increase was mainly observed in the HBV mantle protein stimulation group.

圖 1 ：圖解說明實例性反義寡核苷酸偶聯物，其中將寡核苷酸表示為波浪線(A-D)或「寡核苷酸」 (E-H)或T2 (I)且靶向去唾液酸醣蛋白受體之偶聯物部分係三價N-乙醯基半乳糖胺部分。化合物A至D包括二離胺酸支化劑分子、PEG3間隔體及三個末端GalNAc碳水化合物部分。在化合物A及B中，寡核苷酸無需連接體即直接連接至靶向去唾液酸醣蛋白受體之偶聯物部分。在化合物C及D中，寡核苷酸經由C6連接體直接連接至靶向去唾液酸醣蛋白受體之偶聯物部分。化合物E-I包括三倍增體支化劑分子及具有不同長度及結構之間隔體及三個末端GalNAc碳水化合物部分。圖 2 ：圖形展示實例2中所測試化合物與其在靶核酸上之位置相關之EC50 (A)及PD-L1敲低 (以鹽水%形式) (B)。測試化合物之細胞系係THP1(●)及Karpas (

Figure 02_image001
)。圖 3 ：三價GalNAc簇(GN2)之結構式。GN2可用作本發明中之偶聯物部分。波浪線圖解說明簇至(例如) C6胺基連接體或直接至寡核苷酸之偶聯位點。圖 4 ：CMP ID NO 766_2之結構式。圖 5 ：CMP ID NO 767_2之結構式。圖 6 ：CMP ID NO 768_2之結構式。圖 7 ：CMP ID NO 769_2之結構式。圖 8 ：CMP ID NO 770_2之結構式。圖 9 ：檢測來自在使用鹽水及所指示CMP ID NO治療後之聚(IC)誘導動物之肝中之PD-L1蛋白表現的西方印漬(Western blot)。每一印漬展示裸寡核苷酸與相同寡核苷酸之GalNAc偶聯形式，印漬A) CMP ID NO 744_1及755_2，B)  CMP ID NO 747_1及758_2，C) CMP ID NO 748_1及759_2，D) CMP ID NO 752_1及763_2及E) CMP ID NO 753_1及764_2。上帶係紐蛋白載量對照，下帶係PD-L1蛋白。每一印漬中之第一泳道展示並無聚(IC)誘導之鹽水治療小鼠。該等小鼠表現極少PD-L蛋白。圖 10 ：在使用●媒劑(組10及1)、◆ DNA疫苗(組11及2)、○抗PD-L1抗體(組12)、▲裸PD-L1 ASO + DNA疫苗(組7)或ΔGalNAc偶聯之PD-L1 ASO + DNA疫苗(組8)治療之後肝中之單核細胞之群體，表示每一組之個別動物且藉由每一組之垂直線指示平均值(參見表18)。評價DNA疫苗組與三個治療組之間之統計學顯著性且在存在時其由組間之*指示(* = P＜0.05，*** = P＜ 0.001且**** = P＜0.0001)。A)代表在治療後肝中之T細胞數。B)代表CD4+ T細胞部分且C)代表CD8+ T細胞部分。圖 11 ：在使用●媒劑(組10及1)、◆ DNA疫苗(組11及2)、○抗PD-L1抗體(組12)、▲裸PD-L1 ASO + DNA疫苗(組7)或ΔGalNAc偶聯之PD-L1 ASO + DNA疫苗(組8)治療之後肝中之PD-L1陽性細胞之調節，表示每一組之個別動物且藉由每一組之垂直線指示平均值(參見表19)。評價DNA疫苗組與三個治療組之間之統計學顯著性且在存在時其由組間之*指示(* = P＜0.05且**** = P＜0.0001)。A)代表在治療後肝中之表現PD-L1之CD8+ T細胞之百分比。B)代表在治療後肝中之表現PD-L1之CD4+ T細胞之百分比且C)代表在治療後肝中之表現PD-L1之B細胞之百分比。圖 12 ：在使用●媒劑(組10及1)、◆ DNA疫苗(組11及2)、○抗PD-L1抗體(組12)、▲裸PD-L1 ASO + DNA疫苗(組7)或ΔGalNAc偶聯之PD-L1 ASO + DNA疫苗(組8)治療之後肝中之HBV抗原特異性CD8+細胞介素分泌細胞，表示每一組之個別動物且藉由每一組之垂直線指示平均值(參見表20)。評價DNA疫苗組與三個治療組之間之統計學顯著性且在存在時其由組間之*指示(* = P＜0.05)。A)代表在治療後肝中之HBV PreS2+S抗原特異性IFN-γ分泌性CD8+ T細胞之百分比。B)代表在治療後肝中之HBV核心抗原特異性IFN-γ分泌性CD8+ T細胞之百分比，且C)代表在治療後肝中之HBV PreS2+S抗原特異性IFN-γ及TNF-α分泌性CD8+ T細胞之百分比。圖 13 ：與媒劑(■)相比，在使用GalNAc偶聯之PD-L1反義CMP NO: 759_2 (▼)治療後AAV/HBV小鼠中之HBV-DNA、HBsAg及HBeAg。垂直線指示治療終點。 Figure 1 : Illustrates exemplary antisense oligonucleotide conjugates, where the oligonucleotides are represented as wavy lines (AD) or "oligonucleotides" (EH) or T 2 (I) and targeted to The conjugate part of the sialoglycoprotein receptor is the trivalent N-acetylgalactosamine part. Compounds A to D include a dilysine brancher molecule, a PEG3 spacer, and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotides are directly linked to the conjugate moiety that targets the asialoglycoprotein receptor without a linker. In compounds C and D, the oligonucleotide is directly linked to the conjugate moiety that targets the asialoglycoprotein receptor via a C6 linker. Compound EI includes triploid branching agent molecules, spacers with different lengths and structures, and three terminal GalNAc carbohydrate moieties. Figure 2 : Graphical display of EC50 (A) and PD-L1 knockdown (in saline %) (B) of the compound tested in Example 2 and its position on the target nucleic acid. The cell lines of the test compound were THP1 (●) and Karpas (
Figure 02_image001
). Figure 3 : Structural formula of trivalent GalNAc cluster (GN2). GN2 can be used as the conjugate part in the present invention. The wavy line illustrates the coupling site of the cluster to, for example, a C6 amine linker or directly to the oligonucleotide. Figure 4 : The structure of CMP ID NO 766_2. Figure 5 : The structure of CMP ID NO 767_2. Figure 6 : The structural formula of CMP ID NO 768_2. Figure 7 : The structural formula of CMP ID NO 769_2. Figure 8 : The structural formula of CMP ID NO 770_2. Figure 9 : Western blot to detect PD-L1 protein expression in the liver of animals induced by poly(IC) after treatment with saline and indicated CMP ID NO. Each print shows the GalNAc coupling form of the naked oligonucleotide and the same oligonucleotide, print A) CMP ID NO 744_1 and 755_2, B) CMP ID NO 747_1 and 758_2, C) CMP ID NO 748_1 and 759_2 , D) CMP ID NO 752_1 and 763_2 and E) CMP ID NO 753_1 and 764_2. The upper band is the vinculin load control, and the lower band is the PD-L1 protein. The first lane in each print shows saline-treated mice without poly (IC) induction. These mice showed very little PD-L protein. Figure 10 : In use ● Vehicle (group 10 and 1), ◆ DNA vaccine (group 11 and 2), ○ anti-PD-L1 antibody (group 12), ▲ naked PD-L1 ASO + DNA vaccine (group 7) or The population of monocytes in the liver after treatment with ΔGalNAc-conjugated PD-L1 ASO + DNA vaccine (group 8) represents individual animals in each group and the average value is indicated by the vertical line of each group (see Table 18) . To evaluate the statistical significance between the DNA vaccine group and the three treatment groups and when present, it is indicated by the * between groups (* = P＜0.05, *** = P＜0.001 and **** = P＜0.0001 ). A) represents the number of T cells in the liver after treatment. B) represents the CD4+ T cell fraction and C) represents the CD8+ T cell fraction. Figure 11 : In use ● vehicle (group 10 and 1), ◆ DNA vaccine (group 11 and 2), ○ anti-PD-L1 antibody (group 12), ▲ naked PD-L1 ASO + DNA vaccine (group 7) or The regulation of PD-L1 positive cells in the liver after treatment with ΔGalNAc-conjugated PD-L1 ASO + DNA vaccine (group 8) represents the individual animals of each group and the average value is indicated by the vertical line of each group (see table 19). The statistical significance between the DNA vaccine group and the three treatment groups was evaluated and when present, it was indicated by the * between groups (* = P<0.05 and **** = P<0.0001). A) represents the percentage of CD8+ T cells expressing PD-L1 in the liver after treatment. B) represents the percentage of PD-L1 expressing CD4+ T cells in the liver after treatment and C) represents the percentage of PD-L1 expressing B cells in the liver after treatment. Figure 12 : In use ● vehicle (groups 10 and 1), ◆ DNA vaccine (groups 11 and 2), ○ anti-PD-L1 antibody (group 12), ▲ naked PD-L1 ASO + DNA vaccine (group 7) or ΔGalNAc-conjugated PD-L1 ASO + DNA vaccine (group 8) HBV antigen-specific CD8+ interleukin secreting cells in the liver after treatment, representing individual animals in each group and the average value indicated by the vertical line of each group (See Table 20). The statistical significance between the DNA vaccine group and the three treatment groups was evaluated and when present, it was indicated by the * between groups (* = P<0.05). A) represents the percentage of HBV PreS2+S antigen-specific IFN-γ secreting CD8+ T cells in the liver after treatment. B) represents the percentage of HBV core antigen-specific IFN-γ secreting CD8+ T cells in the liver after treatment, and C) represents the HBV PreS2+S antigen-specific IFN-γ and TNF-α secretion in the liver after treatment Percentage of sex CD8+ T cells. Figure 13 : Compared with vehicle (■), HBV-DNA, HBsAg and HBeAg in AAV/HBV mice after treatment with GalNAc-conjugated PD-L1 antisense CMP NO: 759_2 (▼). The vertical line indicates the end of treatment.

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
Figure 12_A0101_SEQ_0010

Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Figure 12_A0101_SEQ_0014
Figure 12_A0101_SEQ_0014

Figure 12_A0101_SEQ_0015
Figure 12_A0101_SEQ_0015

Figure 12_A0101_SEQ_0016
Figure 12_A0101_SEQ_0016

Figure 12_A0101_SEQ_0017
Figure 12_A0101_SEQ_0017

Figure 12_A0101_SEQ_0018
Figure 12_A0101_SEQ_0018

Figure 12_A0101_SEQ_0019
Figure 12_A0101_SEQ_0019

Figure 12_A0101_SEQ_0020
Figure 12_A0101_SEQ_0020

Figure 12_A0101_SEQ_0021
Figure 12_A0101_SEQ_0021

Figure 12_A0101_SEQ_0022
Figure 12_A0101_SEQ_0022

Figure 12_A0101_SEQ_0023
Figure 12_A0101_SEQ_0023

Figure 12_A0101_SEQ_0024
Figure 12_A0101_SEQ_0024

Figure 12_A0101_SEQ_0025
Figure 12_A0101_SEQ_0025

Figure 12_A0101_SEQ_0026
Figure 12_A0101_SEQ_0026

Figure 12_A0101_SEQ_0027
Figure 12_A0101_SEQ_0027

Figure 12_A0101_SEQ_0028
Figure 12_A0101_SEQ_0028

Figure 12_A0101_SEQ_0029
Figure 12_A0101_SEQ_0029

Figure 12_A0101_SEQ_0030
Figure 12_A0101_SEQ_0030

Figure 12_A0101_SEQ_0031
Figure 12_A0101_SEQ_0031

Figure 12_A0101_SEQ_0032
Figure 12_A0101_SEQ_0032

Figure 12_A0101_SEQ_0033
Figure 12_A0101_SEQ_0033

Figure 12_A0101_SEQ_0034
Figure 12_A0101_SEQ_0034

Figure 12_A0101_SEQ_0035
Figure 12_A0101_SEQ_0035

Figure 12_A0101_SEQ_0036
Figure 12_A0101_SEQ_0036

Figure 12_A0101_SEQ_0037
Figure 12_A0101_SEQ_0037

Figure 12_A0101_SEQ_0038
Figure 12_A0101_SEQ_0038

Figure 12_A0101_SEQ_0039
Figure 12_A0101_SEQ_0039

Figure 12_A0101_SEQ_0040
Figure 12_A0101_SEQ_0040

Figure 12_A0101_SEQ_0041
Figure 12_A0101_SEQ_0041

Figure 12_A0101_SEQ_0042
Figure 12_A0101_SEQ_0042

Figure 12_A0101_SEQ_0043
Figure 12_A0101_SEQ_0043

Figure 12_A0101_SEQ_0044
Figure 12_A0101_SEQ_0044

Figure 12_A0101_SEQ_0045
Figure 12_A0101_SEQ_0045

Figure 12_A0101_SEQ_0046
Figure 12_A0101_SEQ_0046

Figure 12_A0101_SEQ_0047
Figure 12_A0101_SEQ_0047

Figure 12_A0101_SEQ_0048
Figure 12_A0101_SEQ_0048

Figure 12_A0101_SEQ_0049
Figure 12_A0101_SEQ_0049

Figure 12_A0101_SEQ_0050
Figure 12_A0101_SEQ_0050

Figure 12_A0101_SEQ_0051
Figure 12_A0101_SEQ_0051

Figure 12_A0101_SEQ_0052
Figure 12_A0101_SEQ_0052

Figure 12_A0101_SEQ_0053
Figure 12_A0101_SEQ_0053

Figure 12_A0101_SEQ_0054
Figure 12_A0101_SEQ_0054

Figure 12_A0101_SEQ_0055
Figure 12_A0101_SEQ_0055

Figure 12_A0101_SEQ_0056
Figure 12_A0101_SEQ_0056

Figure 12_A0101_SEQ_0057
Figure 12_A0101_SEQ_0057

Figure 12_A0101_SEQ_0058
Figure 12_A0101_SEQ_0058

Figure 12_A0101_SEQ_0059
Figure 12_A0101_SEQ_0059

Figure 12_A0101_SEQ_0060
Figure 12_A0101_SEQ_0060

Figure 12_A0101_SEQ_0061
Figure 12_A0101_SEQ_0061

Figure 12_A0101_SEQ_0062
Figure 12_A0101_SEQ_0062

Figure 12_A0101_SEQ_0063
Figure 12_A0101_SEQ_0063

Figure 12_A0101_SEQ_0064
Figure 12_A0101_SEQ_0064

Figure 12_A0101_SEQ_0065
Figure 12_A0101_SEQ_0065

Figure 12_A0101_SEQ_0066
Figure 12_A0101_SEQ_0066

Figure 12_A0101_SEQ_0067
Figure 12_A0101_SEQ_0067

Figure 12_A0101_SEQ_0068
Figure 12_A0101_SEQ_0068

Figure 12_A0101_SEQ_0069
Figure 12_A0101_SEQ_0069

Figure 12_A0101_SEQ_0070
Figure 12_A0101_SEQ_0070

Figure 12_A0101_SEQ_0071
Figure 12_A0101_SEQ_0071

Figure 12_A0101_SEQ_0072
Figure 12_A0101_SEQ_0072

Figure 12_A0101_SEQ_0073
Figure 12_A0101_SEQ_0073

Figure 12_A0101_SEQ_0074
Figure 12_A0101_SEQ_0074

Figure 12_A0101_SEQ_0075
Figure 12_A0101_SEQ_0075

Figure 12_A0101_SEQ_0076
Figure 12_A0101_SEQ_0076

Figure 12_A0101_SEQ_0077
Figure 12_A0101_SEQ_0077

Figure 12_A0101_SEQ_0078
Figure 12_A0101_SEQ_0078

Figure 12_A0101_SEQ_0079
Figure 12_A0101_SEQ_0079

Figure 12_A0101_SEQ_0080
Figure 12_A0101_SEQ_0080

Figure 12_A0101_SEQ_0081
Figure 12_A0101_SEQ_0081

Figure 12_A0101_SEQ_0082
Figure 12_A0101_SEQ_0082

Figure 12_A0101_SEQ_0083
Figure 12_A0101_SEQ_0083

Figure 12_A0101_SEQ_0084
Figure 12_A0101_SEQ_0084

Figure 12_A0101_SEQ_0085
Figure 12_A0101_SEQ_0085

Figure 12_A0101_SEQ_0086
Figure 12_A0101_SEQ_0086

Figure 12_A0101_SEQ_0087
Figure 12_A0101_SEQ_0087

Figure 12_A0101_SEQ_0088
Figure 12_A0101_SEQ_0088

Figure 12_A0101_SEQ_0089
Figure 12_A0101_SEQ_0089

Figure 12_A0101_SEQ_0090
Figure 12_A0101_SEQ_0090

Figure 12_A0101_SEQ_0091
Figure 12_A0101_SEQ_0091

Figure 12_A0101_SEQ_0092
Figure 12_A0101_SEQ_0092

Figure 12_A0101_SEQ_0093
Figure 12_A0101_SEQ_0093

Figure 12_A0101_SEQ_0094
Figure 12_A0101_SEQ_0094

Figure 12_A0101_SEQ_0095
Figure 12_A0101_SEQ_0095

Figure 12_A0101_SEQ_0096
Figure 12_A0101_SEQ_0096

Figure 12_A0101_SEQ_0097
Figure 12_A0101_SEQ_0097

Figure 12_A0101_SEQ_0098
Figure 12_A0101_SEQ_0098

Figure 12_A0101_SEQ_0099
Figure 12_A0101_SEQ_0099

Figure 12_A0101_SEQ_0100
Figure 12_A0101_SEQ_0100

Figure 12_A0101_SEQ_0101
Figure 12_A0101_SEQ_0101

Figure 12_A0101_SEQ_0102
Figure 12_A0101_SEQ_0102

Figure 12_A0101_SEQ_0103
Figure 12_A0101_SEQ_0103

Figure 12_A0101_SEQ_0104
Figure 12_A0101_SEQ_0104

Figure 12_A0101_SEQ_0105
Figure 12_A0101_SEQ_0105

Figure 12_A0101_SEQ_0106
Figure 12_A0101_SEQ_0106

Figure 12_A0101_SEQ_0107
Figure 12_A0101_SEQ_0107

Figure 12_A0101_SEQ_0108
Figure 12_A0101_SEQ_0108

Figure 12_A0101_SEQ_0109
Figure 12_A0101_SEQ_0109

Figure 12_A0101_SEQ_0110
Figure 12_A0101_SEQ_0110

Figure 12_A0101_SEQ_0111
Figure 12_A0101_SEQ_0111

Figure 12_A0101_SEQ_0112
Figure 12_A0101_SEQ_0112

Figure 12_A0101_SEQ_0113
Figure 12_A0101_SEQ_0113

Figure 12_A0101_SEQ_0114
Figure 12_A0101_SEQ_0114

Figure 12_A0101_SEQ_0115
Figure 12_A0101_SEQ_0115

Figure 12_A0101_SEQ_0116
Figure 12_A0101_SEQ_0116

Figure 12_A0101_SEQ_0117
Figure 12_A0101_SEQ_0117

Figure 12_A0101_SEQ_0118
Figure 12_A0101_SEQ_0118

Figure 12_A0101_SEQ_0119
Figure 12_A0101_SEQ_0119

Figure 12_A0101_SEQ_0120
Figure 12_A0101_SEQ_0120

Figure 12_A0101_SEQ_0121
Figure 12_A0101_SEQ_0121

Figure 12_A0101_SEQ_0122
Figure 12_A0101_SEQ_0122

Figure 12_A0101_SEQ_0123
Figure 12_A0101_SEQ_0123

Figure 12_A0101_SEQ_0124
Figure 12_A0101_SEQ_0124

Figure 12_A0101_SEQ_0125
Figure 12_A0101_SEQ_0125

Figure 12_A0101_SEQ_0126
Figure 12_A0101_SEQ_0126

Figure 12_A0101_SEQ_0127
Figure 12_A0101_SEQ_0127

Figure 12_A0101_SEQ_0128
Figure 12_A0101_SEQ_0128

Figure 12_A0101_SEQ_0129
Figure 12_A0101_SEQ_0129

Figure 12_A0101_SEQ_0130
Figure 12_A0101_SEQ_0130

Figure 12_A0101_SEQ_0131
Figure 12_A0101_SEQ_0131

Figure 12_A0101_SEQ_0132
Figure 12_A0101_SEQ_0132

Figure 12_A0101_SEQ_0133
Figure 12_A0101_SEQ_0133

Figure 12_A0101_SEQ_0134
Figure 12_A0101_SEQ_0134

Figure 12_A0101_SEQ_0135
Figure 12_A0101_SEQ_0135

Figure 12_A0101_SEQ_0136
Figure 12_A0101_SEQ_0136

Figure 12_A0101_SEQ_0137
Figure 12_A0101_SEQ_0137

Figure 12_A0101_SEQ_0138
Figure 12_A0101_SEQ_0138

Figure 12_A0101_SEQ_0139
Figure 12_A0101_SEQ_0139

Figure 12_A0101_SEQ_0140
Figure 12_A0101_SEQ_0140

Figure 12_A0101_SEQ_0141
Figure 12_A0101_SEQ_0141

Figure 12_A0101_SEQ_0142
Figure 12_A0101_SEQ_0142

Figure 12_A0101_SEQ_0143
Figure 12_A0101_SEQ_0143

Figure 12_A0101_SEQ_0144
Figure 12_A0101_SEQ_0144

Figure 12_A0101_SEQ_0145
Figure 12_A0101_SEQ_0145

Figure 12_A0101_SEQ_0146
Figure 12_A0101_SEQ_0146

Figure 12_A0101_SEQ_0147
Figure 12_A0101_SEQ_0147

Figure 12_A0101_SEQ_0148
Figure 12_A0101_SEQ_0148

Figure 12_A0101_SEQ_0149
Figure 12_A0101_SEQ_0149

Figure 12_A0101_SEQ_0150
Figure 12_A0101_SEQ_0150

Figure 12_A0101_SEQ_0151
Figure 12_A0101_SEQ_0151

Figure 12_A0101_SEQ_0152
Figure 12_A0101_SEQ_0152

Figure 12_A0101_SEQ_0153
Figure 12_A0101_SEQ_0153

Figure 12_A0101_SEQ_0154
Figure 12_A0101_SEQ_0154

Figure 12_A0101_SEQ_0155
Figure 12_A0101_SEQ_0155

Figure 12_A0101_SEQ_0156
Figure 12_A0101_SEQ_0156

Figure 12_A0101_SEQ_0157
Figure 12_A0101_SEQ_0157

Figure 12_A0101_SEQ_0158
Figure 12_A0101_SEQ_0158

Figure 12_A0101_SEQ_0159
Figure 12_A0101_SEQ_0159

Figure 12_A0101_SEQ_0160
Figure 12_A0101_SEQ_0160

Figure 12_A0101_SEQ_0161
Figure 12_A0101_SEQ_0161

Figure 12_A0101_SEQ_0162
Figure 12_A0101_SEQ_0162

Figure 12_A0101_SEQ_0163
Figure 12_A0101_SEQ_0163

Figure 12_A0101_SEQ_0164
Figure 12_A0101_SEQ_0164

Figure 12_A0101_SEQ_0165
Figure 12_A0101_SEQ_0165

Figure 12_A0101_SEQ_0166
Figure 12_A0101_SEQ_0166

Figure 12_A0101_SEQ_0167
Figure 12_A0101_SEQ_0167

Figure 12_A0101_SEQ_0168
Figure 12_A0101_SEQ_0168

Figure 12_A0101_SEQ_0169
Figure 12_A0101_SEQ_0169

Figure 12_A0101_SEQ_0170
Figure 12_A0101_SEQ_0170

Figure 12_A0101_SEQ_0171
Figure 12_A0101_SEQ_0171

Figure 12_A0101_SEQ_0172
Figure 12_A0101_SEQ_0172

Figure 12_A0101_SEQ_0173
Figure 12_A0101_SEQ_0173

Figure 12_A0101_SEQ_0174
Figure 12_A0101_SEQ_0174

Figure 12_A0101_SEQ_0175
Figure 12_A0101_SEQ_0175

Figure 12_A0101_SEQ_0176
Figure 12_A0101_SEQ_0176

Figure 12_A0101_SEQ_0177
Figure 12_A0101_SEQ_0177

Figure 12_A0101_SEQ_0178
Figure 12_A0101_SEQ_0178

Figure 12_A0101_SEQ_0179
Figure 12_A0101_SEQ_0179

Figure 12_A0101_SEQ_0180
Figure 12_A0101_SEQ_0180

Figure 12_A0101_SEQ_0181
Figure 12_A0101_SEQ_0181

Figure 12_A0101_SEQ_0182
Figure 12_A0101_SEQ_0182

Figure 12_A0101_SEQ_0183
Figure 12_A0101_SEQ_0183

Figure 12_A0101_SEQ_0184
Figure 12_A0101_SEQ_0184

Figure 12_A0101_SEQ_0185
Figure 12_A0101_SEQ_0185

Figure 12_A0101_SEQ_0186
Figure 12_A0101_SEQ_0186

Figure 12_A0101_SEQ_0187
Figure 12_A0101_SEQ_0187

Figure 12_A0101_SEQ_0188
Figure 12_A0101_SEQ_0188

Figure 12_A0101_SEQ_0189
Figure 12_A0101_SEQ_0189

Figure 12_A0101_SEQ_0190
Figure 12_A0101_SEQ_0190

Figure 12_A0101_SEQ_0191
Figure 12_A0101_SEQ_0191

Figure 12_A0101_SEQ_0192
Figure 12_A0101_SEQ_0192

Figure 12_A0101_SEQ_0193
Figure 12_A0101_SEQ_0193

Figure 12_A0101_SEQ_0194
Figure 12_A0101_SEQ_0194

Figure 12_A0101_SEQ_0195
Figure 12_A0101_SEQ_0195

Figure 12_A0101_SEQ_0196
Figure 12_A0101_SEQ_0196

Figure 12_A0101_SEQ_0197
Figure 12_A0101_SEQ_0197

Figure 12_A0101_SEQ_0198
Figure 12_A0101_SEQ_0198

Figure 12_A0101_SEQ_0199
Figure 12_A0101_SEQ_0199

Figure 12_A0101_SEQ_0200
Figure 12_A0101_SEQ_0200

Figure 12_A0101_SEQ_0201
Figure 12_A0101_SEQ_0201

Figure 12_A0101_SEQ_0202
Figure 12_A0101_SEQ_0202

Figure 12_A0101_SEQ_0203
Figure 12_A0101_SEQ_0203

Figure 12_A0101_SEQ_0204
Figure 12_A0101_SEQ_0204

Figure 12_A0101_SEQ_0205
Figure 12_A0101_SEQ_0205

Figure 12_A0101_SEQ_0206
Figure 12_A0101_SEQ_0206

Figure 12_A0101_SEQ_0207
Figure 12_A0101_SEQ_0207

Figure 12_A0101_SEQ_0208
Figure 12_A0101_SEQ_0208

Figure 12_A0101_SEQ_0209
Figure 12_A0101_SEQ_0209

Figure 12_A0101_SEQ_0210
Figure 12_A0101_SEQ_0210

Figure 12_A0101_SEQ_0211
Figure 12_A0101_SEQ_0211

Figure 12_A0101_SEQ_0212
Figure 12_A0101_SEQ_0212

Figure 12_A0101_SEQ_0213
Figure 12_A0101_SEQ_0213

Figure 12_A0101_SEQ_0214
Figure 12_A0101_SEQ_0214

Figure 12_A0101_SEQ_0215
Figure 12_A0101_SEQ_0215

Figure 12_A0101_SEQ_0216
Figure 12_A0101_SEQ_0216

Figure 12_A0101_SEQ_0217
Figure 12_A0101_SEQ_0217

Figure 12_A0101_SEQ_0218
Figure 12_A0101_SEQ_0218

Figure 12_A0101_SEQ_0219
Figure 12_A0101_SEQ_0219

Figure 12_A0101_SEQ_0220
Figure 12_A0101_SEQ_0220

Figure 12_A0101_SEQ_0221
Figure 12_A0101_SEQ_0221

Figure 12_A0101_SEQ_0222
Figure 12_A0101_SEQ_0222

Figure 12_A0101_SEQ_0223
Figure 12_A0101_SEQ_0223

Figure 12_A0101_SEQ_0224
Figure 12_A0101_SEQ_0224

Figure 12_A0101_SEQ_0225
Figure 12_A0101_SEQ_0225

Figure 12_A0101_SEQ_0226
Figure 12_A0101_SEQ_0226

Figure 12_A0101_SEQ_0227
Figure 12_A0101_SEQ_0227

Figure 12_A0101_SEQ_0228
Figure 12_A0101_SEQ_0228

Figure 12_A0101_SEQ_0229
Figure 12_A0101_SEQ_0229

Figure 12_A0101_SEQ_0230
Figure 12_A0101_SEQ_0230

Figure 12_A0101_SEQ_0231
Figure 12_A0101_SEQ_0231

Figure 12_A0101_SEQ_0232
Figure 12_A0101_SEQ_0232

Figure 12_A0101_SEQ_0233
Figure 12_A0101_SEQ_0233

Figure 12_A0101_SEQ_0234
Figure 12_A0101_SEQ_0234

Figure 12_A0101_SEQ_0235
Figure 12_A0101_SEQ_0235

Figure 12_A0101_SEQ_0236
Figure 12_A0101_SEQ_0236

Figure 12_A0101_SEQ_0237
Figure 12_A0101_SEQ_0237

Figure 12_A0101_SEQ_0238
Figure 12_A0101_SEQ_0238

Figure 12_A0101_SEQ_0239
Figure 12_A0101_SEQ_0239

Figure 12_A0101_SEQ_0240
Figure 12_A0101_SEQ_0240

Figure 12_A0101_SEQ_0241
Figure 12_A0101_SEQ_0241

Figure 12_A0101_SEQ_0242
Figure 12_A0101_SEQ_0242

Figure 12_A0101_SEQ_0243
Figure 12_A0101_SEQ_0243

Figure 12_A0101_SEQ_0244
Figure 12_A0101_SEQ_0244

Figure 12_A0101_SEQ_0245
Figure 12_A0101_SEQ_0245

Figure 12_A0101_SEQ_0246
Figure 12_A0101_SEQ_0246

Claims (32)

一種反義寡核苷酸偶聯物(conjugate)，其包含： a. 寡核苷酸，其包括與PD-L1靶核酸具有至少90%互補性之長度10至30個核苷酸之鄰接核苷酸序列；及 b. 至少一個靶向去唾液酸醣蛋白受體之偶聯物部分，其共價連接至a)中之寡核苷酸，其中該鄰接核苷酸序列與該靶核酸之子序列互補，其中該子序列為SEQ ID NO: 1上之位置5467至12107。An antisense oligonucleotide conjugate, which comprises: a. Oligonucleotides, which include adjacent nucleotide sequences of 10 to 30 nucleotides in length with at least 90% complementarity with the PD-L1 target nucleic acid; and b. At least one conjugate portion targeting an asialoglycoprotein receptor, which is covalently linked to the oligonucleotide in a), wherein the adjacent nucleotide sequence is complementary to a subsequence of the target nucleic acid, wherein the The subsequence is positions 5467 to 12107 on SEQ ID NO:1. 如請求項1之反義寡核苷酸偶聯物，其中該寡核苷酸包含SEQ ID NO: 466之序列。The antisense oligonucleotide conjugate of claim 1, wherein the oligonucleotide comprises the sequence of SEQ ID NO: 466. 如請求項1或2之反義寡核苷酸偶聯物，其中該鄰接核苷酸序列包含一或多個經修飾核苷。The antisense oligonucleotide conjugate of claim 1 or 2, wherein the adjacent nucleotide sequence comprises one or more modified nucleosides. 如請求項3之反義寡核苷酸偶聯物，其中該一或多個經修飾核苷為2’糖修飾性核苷。The antisense oligonucleotide conjugate of claim 3, wherein the one or more modified nucleosides are 2'sugar modified nucleosides. 如請求項4之反義寡核苷酸偶聯物，其中該一或多個2’糖修飾性核苷獨立地選自由以下組成之群：2’-O-烷基-RNA、2’-O-甲基-RNA、2’-烷氧基-RNA、2’-O-甲氧基乙基-RNA、2’-胺基-DNA、2’-氟-DNA、阿拉伯糖核酸(ANA)、2’-氟-ANA及鎖核酸(LNA)核苷。The antisense oligonucleotide conjugate of claim 4, wherein the one or more 2'sugar-modified nucleosides are independently selected from the group consisting of 2'-O-alkyl-RNA, 2'- O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA) , 2'-fluoro-ANA and locked nucleic acid (LNA) nucleosides. 如請求項3之反義寡核苷酸偶聯物，其中所有該等經修飾核苷皆為LNA核苷。Such as the antisense oligonucleotide conjugate of claim 3, wherein all the modified nucleosides are LNA nucleosides. 如請求項1或2之反義寡核苷酸偶聯物，其中該鄰接核苷酸序列包含至少一個經修飾核苷間鏈接。The antisense oligonucleotide conjugate of claim 1 or 2, wherein the adjacent nucleotide sequence comprises at least one modified internucleoside linkage. 如請求項7之反義寡核苷酸偶聯物，其中該至少一個經修飾核苷間鏈接為硫代磷酸酯核苷間鏈接。The antisense oligonucleotide conjugate of claim 7, wherein the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage. 如請求項1或2之反義寡核苷酸偶聯物，其中該寡核苷酸為間隙聚體(gapmer)。The antisense oligonucleotide conjugate of claim 1 or 2, wherein the oligonucleotide is a gapmer. 如請求項1或2之反義寡核苷酸偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分包含至少一個選自由以下組成之群之碳水化合物部分：半乳糖、半乳糖胺、N-甲醯基-半乳糖胺、N-乙醯基半乳糖胺、N-丙醯基-半乳糖胺、N-正丁醯基-半乳糖胺及N-異丁醯基半乳糖胺。The antisense oligonucleotide conjugate of claim 1 or 2, wherein the conjugate portion targeted to the asialoglycoprotein receptor comprises at least one carbohydrate portion selected from the group consisting of galactose, Galactosamine, N-methanyl-galactosamine, N-acetylgalactosamine, N-propanyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl-galactosamine. 如請求項1或2之反義寡核苷酸偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分為單價、二價、三價或四價。The antisense oligonucleotide conjugate of claim 1 or 2, wherein the conjugate portion of the asialoglycoprotein receptor-targeting conjugate is monovalent, bivalent, trivalent or tetravalent. 如請求項1或2之反義寡核苷酸偶聯物，其中該靶向去唾液酸醣蛋白受體之偶聯物部分為三價N-乙醯基半乳糖胺(GalNAc)部分。The antisense oligonucleotide conjugate according to claim 1 or 2, wherein the conjugate moiety targeted to the asialoglycoprotein receptor is a trivalent N-acetylgalactosamine (GalNAc) moiety. 一種具式CTAattgtagtagtaCTC之反義寡核苷酸，其中大寫字母代表β-D-氧基鎖核酸(LNA)核苷，小寫字母代表DNA核苷，所有LNA C皆為5-甲基胞嘧啶，且所有核苷間鏈接皆為硫代磷酸酯核苷間鏈接。A kind of antisense oligonucleotide with CTAattgtagtagtaCTC, in which the uppercase letters represent β-D-oxygen-locked nucleic acid (LNA) nucleosides, the lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosines, and All internucleoside linkages are phosphorothioate internucleoside linkages. 一種反義寡核苷酸偶聯物，其包含如請求項13之寡核苷酸及共價連接至該寡核苷酸之偶聯物部分。An antisense oligonucleotide conjugate comprising the oligonucleotide as claimed in claim 13 and the conjugate part covalently linked to the oligonucleotide. 如請求項14之反義寡核苷酸偶聯物，其中連接體存在於該寡核苷酸與該偶聯物部分之間。The antisense oligonucleotide conjugate of claim 14, wherein a linker exists between the oligonucleotide and the conjugate part. 如請求項14或15之反義寡核苷酸偶聯物，其中該偶聯物部分為去唾液酸醣蛋白受體靶向部分。The antisense oligonucleotide conjugate of claim 14 or 15, wherein the conjugate part is an asialoglycoprotein receptor targeting part. 如請求項16之反義寡核苷酸偶聯物，其中該去唾液酸醣蛋白受體靶向部分為三價N-乙醯基半乳糖胺(GalNAc)部分。The antisense oligonucleotide conjugate according to claim 16, wherein the asialoglycoprotein receptor targeting moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety. 如請求項15之反義寡核苷酸偶聯物，其中該連接體為生理上不穩定連接體。The antisense oligonucleotide conjugate of claim 15, wherein the linker is a physiologically unstable linker. 如請求項18之反義寡核苷酸偶聯物，其中該生理上不穩定連接體為核酸酶易感連接體。The antisense oligonucleotide conjugate of claim 18, wherein the physiologically unstable linker is a nuclease susceptible linker. 如請求項18或19之反義寡核苷酸偶聯物，其中該生理上不穩定連接體包含胞苷-腺苷二核苷酸。The antisense oligonucleotide conjugate of claim 18 or 19, wherein the physiologically unstable linker comprises cytidine-adenosine dinucleotide. 如請求項14之反義寡核苷酸偶聯物，其中連接體存在於該寡核苷酸與該偶聯物部分之間；進一步其中該偶聯物部分為去唾液酸醣蛋白受體靶向部分，其為三價N-乙醯基半乳糖胺(GalNAc)部分；其中該連接體為生理上不穩定連接體；進一步其中該生理上不穩定連接體包含胞苷-腺苷二核苷酸。The antisense oligonucleotide conjugate of claim 14, wherein a linker exists between the oligonucleotide and the conjugate part; further wherein the conjugate part is an asialoglycoprotein receptor target A part, which is a trivalent N-acetylgalactosamine (GalNAc) part; wherein the linker is a physiologically unstable linker; further wherein the physiologically unstable linker comprises cytidine-adenosine dinucleoside acid. 一種醫藥組合物，其包括如請求項1至12及14至21中任一項之反義寡核苷酸偶聯物或如請求項13之反義寡核苷酸及醫藥上可接受之稀釋劑、溶劑、載劑、鹽及/或佐劑。A pharmaceutical composition comprising the antisense oligonucleotide conjugate according to any one of claims 1 to 12 and 14 to 21 or the antisense oligonucleotide according to claim 13 and a pharmaceutically acceptable dilution Agents, solvents, carriers, salts and/or adjuvants. 如請求項22之醫藥組合物，其中該醫藥上可接受之稀釋劑為無菌磷酸鹽緩衝鹽水。The pharmaceutical composition of claim 22, wherein the pharmaceutically acceptable diluent is sterile phosphate buffered saline. 如請求項22或23之醫藥組合物，其中該醫藥上可接受之鹽為鈉鹽。The pharmaceutical composition of claim 22 or 23, wherein the pharmaceutically acceptable salt is a sodium salt. 如請求項22或23之醫藥組合物，其中該醫藥上可接受之鹽為鉀鹽。The pharmaceutical composition of claim 22 or 23, wherein the pharmaceutically acceptable salt is a potassium salt. 一種調節表現PD-L1之靶細胞中PD-L1表現之活體外方法，該方法包括向該細胞投與有效量之如請求項1至12及14至21中任一項之反義寡核苷酸偶聯物、如請求項13之反義寡核苷酸或如請求項22至25中任一項之醫藥組合物。An in vitro method for regulating PD-L1 expression in target cells expressing PD-L1, the method comprising administering to the cell an effective amount of an antisense oligonucleotide such as any one of claims 1 to 12 and 14 to 21 An acid conjugate, an antisense oligonucleotide as claimed in claim 13, or a pharmaceutical composition as claimed in any one of claims 22 to 25. 一種如請求項1至12及14至21中任一項之反義寡核苷酸偶聯物、如請求項13之反義寡核苷酸或如請求項22至25中任一項之醫藥組合物之用途，其用以製備用於調節表現PD-L1之靶細胞中PD-L1表現之醫藥品。An antisense oligonucleotide conjugate as claimed in any one of claims 1 to 12 and 14 to 21, an antisense oligonucleotide as claimed in claim 13 or a medicine as claimed in any one of claims 22 to 25 The purpose of the composition is to prepare pharmaceuticals for regulating the expression of PD-L1 in target cells expressing PD-L1. 一種如請求項1至12及14至21中任一項之反義寡核苷酸偶聯物、如請求項13之反義寡核苷酸或如請求項22至25中任一項之醫藥組合物之用途，其用以製備用於恢復針對病毒之免疫反應之醫藥品。An antisense oligonucleotide conjugate as claimed in any one of claims 1 to 12 and 14 to 21, an antisense oligonucleotide as claimed in claim 13 or a medicine as claimed in any one of claims 22 to 25 The use of the composition is to prepare medicines for restoring the immune response against viruses. 如請求項28之用途，其中該病毒為HBV。Such as the use of claim 28, wherein the virus is HBV. 一種如請求項1至12及14至21中任一項之反義寡核苷酸偶聯物、如請求項13之反義寡核苷酸或如請求項22至25中任一項之醫藥組合物之用途，其用以製備用於恢復針對寄生蟲之免疫反應之醫藥品。An antisense oligonucleotide conjugate as claimed in any one of claims 1 to 12 and 14 to 21, an antisense oligonucleotide as claimed in claim 13 or a medicine as claimed in any one of claims 22 to 25 The purpose of the composition is to prepare medicines for restoring the immune response against parasites. 如請求項28至30中任一項之用途，其中該免疫反應之恢復係在與對照相比時肝中特異性針對一或多種HBV抗原之CD8+ T細胞增加。The use according to any one of claims 28 to 30, wherein the recovery of the immune response is an increase in CD8+ T cells specific for one or more HBV antigens in the liver when compared with the control. 一種如請求項1至12及14至21中任一項之反義寡核苷酸偶聯物、如請求項13之反義寡核苷酸或如請求項22至25中任一項之醫藥組合物之用途，其用以製備用於治療或預防HBV感染之醫藥品。An antisense oligonucleotide conjugate as claimed in any one of claims 1 to 12 and 14 to 21, an antisense oligonucleotide as claimed in claim 13 or a medicine as claimed in any one of claims 22 to 25 The purpose of the composition is to prepare medicines for the treatment or prevention of HBV infection.
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